Inhibition of Apoptosis-Specific eIF-5A(elF-5A1&#34;) with Antisense Oligonucleotides and siRNA as Anti-Inflammatory Therapeutics

ABSTRACT

The present invention relates to apoptosis specific eucaryotic initiation factor 5A (eIF-5A), referred to as apoptosis-specific eIF-5A or eIF5A1, nucleic acids and polypeptides and methods for inhibiting or suppressing apoptosis in cells using antisense nucleotides or siRNAs to inhibit expression of apoptosis-specific eIF-5A. The invention also relates to suppressing or inhibiting expression of pro-inflammatory cytokines or inhibiting activation of NFkB by inhibiting expression of apoptosis-specific eIF-5A.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/383,614, filed on Mar. 10, 2003, which is a continuation-in-part of10/277,969, filed Oct. 23, 2002, which is a continuation-in-part of10/200,148, filed on Jul. 23, 2002, which is a continuation-in-part ofU.S. application Ser. No. 10/141,647, filed May 7, 2002, which is acontinuation-in part of U.S. application Ser. No. 9/909,796, filed Jul.23, 2001, all of which are herein incorporated in their entirety. Thisapplication also claims priority to U.S. provisional 60/476,194 filed onJun. 6, 2003; U.S. provisional 60/504,731 filed on Sep. 22, 2003; U.S.provisional 60/528,249 filed on Dec. 10, 2003; U.S. provisional60/557,671 filed on Mar. 21, 2004 and U.S. provisional 60/(awaited)filed on Jun. 2, 2004, all of which are herein incorporated in theirentirety.

FIELD OF THE INVENTION

The present invention relates to apoptosis-specific eucaryoticinitiation factor (“eIF-5A”) or referred to as “apoptosis-specificeIF-5A” or“eIF-5A 1” and deoxyhypusine synthase (DHS). The presentinvention relates to apoptosis-specific eIF-5A and DHS nucleic acids andpolypeptides and methods for inhibiting expression of apoptosis-specificeIF-5A and DHS.

BACKGROUND OF THE INVENTION

Apoptosis is a genetically programmed cellular event that ischaracterized by well-defined morphological features, such as cellshrinkage, chromatin condensation, nuclear fragmentation, and membraneblebbing. Kerr et al. (1972) Br. J. Cancer, 26, 239-257; Wylie et al.(1980) hit. Rev. Cytol., 68, 251-306. It plays an important role innormal tissue development and homeostasis, and defects in the apoptoticprogram are thought to contribute to a wide range of human disordersranging from neurodegenera and autoimmunity disorders to neoplasms.Thompson (1995) Science, 267, 1456-1462; Mullauer et al. (2001) Mutat.Res, 488, 211-231. Although the morphological characteristics ofapoptotic cells are well characterized, the molecular pathways thatregulate this process have only begun to be elucidated.

One group of proteins that is thought to play a key role in apoptosis isa family of cysteine proteases, termed caspases, which appear to berequired for most pathways of apoptosis. Creagh & Martin (2001) Biochem.Soc. Trans, 29, 696-701; Dales et al. (2001) Leuk. Lymphoma, 41,247-253. Caspases trigger apoptosis in response to apoptotic stimuli bycleaving various cellular proteins, which results in classicmanifestations of apoptosis, including cell shrinkage, membrane blebbingand DNA fragmentation. Chang &. Yang (2000) Microbiol. Mol. Biol. Rev.,64, 821-846.

Pro-apoptotic proteins, such as Bax or Bak, also play a key role in theapoptotic pathway by releasing caspase-activating molecules, such asmitochondrial cytochrome c, thereby promoting cell death throughapoptosis. Martinou & Green (2001) Nat. Rev. Mol. Cell. Biol., 2, 63-67;Zou et al. (1997) Cell, 90, 405-413. Anti-apoptotic proteins, such asBcl-2, promote cell survival by antagonizing the activity of thepro-apoptotic proteins, Bax and Bak. Tsujimoto (1998) Genes Cells, 3,697-707; Kroemer (1997) Nature Med., 3, 614-620. The ratio of Bax:Bcl-2is thought to be one way in which cell fate is determined; an excess ofBax promotes apoptosis and an excess of Bcl-2 promotes cell survival.Salomons et al. (1997) Int. J. Cancer, 71, 959-965; Wallace-Brodeur &Lowe (1999) Cell Mol. Life. Sci., 55, 64-75.

Another key protein involved in apoptosis is a protein that encoded bythe tumor suppressor gene p53. This protein is a transcription factorthat regulates cell growth and induces apoptosis in cells that aredamaged and genetically unstable, presumably through up-regulation ofBax. Bold et al. (1997) Surgical Oncology, 6, 133-142; Ronen et al.,1996; Schuler & Green (2001) Biochem. Soc. Trans., 29, 684-688; Ryan etal. (2001) Curr. Opin. Cell Biol., 13, 332-337; Zömig et al. (2001)Biochem. Biophys. Acta, 1551, F1-F37.

Alterations in the apoptotic pathways are believed to play a key role ina number of disease processes, including cancer. Wyllie et al. (1980)Int. Rev. Cytol., 68, 251-306; Thompson (1995) Science, 267, 1456-1462;Sen & D'Incalci (1992) FEBS Letters, 307, 122-127; McDonnell et al.(1995) Seminars in Cancer and Biology, 6, 53-60. Investigations intocancer development and progression have traditionally been focused oncellular proliferation. However, the important role that apoptosis playsin tumorigenesis has recently become apparent. In fact, much of what isnow known about apoptosis has been learned using tumor models, since thecontrol of apoptosis is invariably altered in some way in tumor cells.Bold et al. (1997) Surgical Oncology, 133-142.

Cytokines also have been implicated in the apoptotic pathway. Biologicalsystems require cellular interactions for their regulation, andcross-talk between cells generally involves a large variety ofcytokines. Cytokines are mediators that are produced in response to awide variety of stimuli by many different cell types. Cytokines arepleiotropic molecules that can exert many different effects on manydifferent cell types, but are especially important in regulation of theimmune response and hematopoietic cell proliferation anddifferentiation. The actions of cytokines on target cells can promotecell survival, proliferation, activation, differentiation, or apoptosisdepending on the particular cytokine, relative concentration, andpresence of other mediators.

The use of anti-cytokines to treat autoimmune disorders such aspsoriasis, rheumatoid arthritis, and Crohn's disease is gainingpopularity. The pro-inflammatory cytokines IL-1 and TNT play a largerole in the pathology of these chronic disorders. Anti-cytokinetherapies that reduce the biological activities of these two cytokinescan provide therapeutic benefits (Dinarello and Abraham, 2002).

Interleukin 1 (IL-1) is an important cytokine that mediates local andsystemic inflammatory reactions and which can synergize with TNF in thepathogenesis of many disorders, including vasculitis, osteoporosis,neurodegenerative disorders, diabetes, lupus nephritis, and autoimmunedisorders such as rheumatoid arthritis. The importance of IL-1β intumour angiogenesis and invasiveness was also recently demonstrated bythe resistance of IL-1β knockout mice to metastases and angiogenesiswhen injected with melanoma cells (Voronov et al., 2003).

Interleukin 18 (IL-18) is a recently discovered member of the IL-1family and is related by structure, receptors, and function to IL-1.IL-18 is a central cytokine involved in inflammatory and autoimmunedisorders as a result of its ability to induce interferon-gamma (IFN-γ),TNF-α, and IL-1. IL-1β and IL-18 are both Capable of inducing productionof TNF-α, a cytokine known to contribute to cardiac dysfunction duringmyocardial ischemia (Maekawa et al., 2002). Inhibition of IL-18 byneutralization with an IL-18 binding protein was found to reduceischemia-induced myocardial dysfunction in an ischemiaireperfusion modelof suprafused human atrial myocardium (Dinarello, 2001). Neutralizationof IL-18 using a mouse IL-18 binding protein was also able to decreaseIFN-γ, TNF-α, and IL-1β transcript levels and reduce joint damage in acollagen-induced arthritis mouse model (Banda et al., 2003). A reductionof IL-18 production or availability may also prove beneficial to controlmetastatic cancer as injection of IL-18 binding protein in a mousemelanoma model successfully inhibited metastases (Carrascal et al.,2003). As a further indication of its importance as a pro-inflammatorycytokine, plasma levels of IL-18 were elevated in patients with chronicliver disease and increased levels were correlated with the severity ofthe disease (Ludwiczek et al., 2002). Similarly, IL-18 and TNF-α wereelevated in the serum of diabetes mellitus patients with nephropathy(Moriwaki et al., 2003). Neuroinflammation following traumatic braininjury is also mediated by pro-inflammatory cytokines and inhibition ofIL-18 by the IL-18 binding protein improved neurological recovery inmice following brain trauma (Yatsiv et al., 2002).

TNF-α, a member of the TNF family of cytokines, is a pro-inflammatorycytokine with pleiotropic effects ranging from co-mitogenic effects onhematopoietic cells, induction of inflammatory responses, and inductionof cell death in many cell types. TNF-α is normally induced by bacteriallipopolysaccharides, parasites, viruses, malignant cells and cytokinesand usually acts beneficially to protect cells from infection andcancer. However, inappropriate induction of TNF-α is a major contributorto disorders resulting from acute and chronic inflammation such asautoimmune disorders and can also contribute to cancer, AIDS, heartdisease, and sepsis (reviewed by Aggarwal and Natarajan, 1996; Sharmaand Anker, 2002). Experimental animal models of disease (i.e. septicshock and rheumatoid arthritis) as well as human disorders (i.e.inflammatory bowel diseases and acute graft-versus-host disease) havedemonstrated the beneficial effects of blocking TNF-α (Wallach et al.,1999). Inhibition of TNF-α has also been effective in providing reliefto patients suffering autoimmune disorders such as Crohn's disease (vanDeventer, 1999) and rheumatoid arthritis (Richard-Miceli and Dougados,2001). The ability of TNF-α to promote the survival and growth of Blymphocytes is also thought to play a role in the pathogenesis of B-cellchronic lymphocytic leukemia (B-CLL) and the levels of TNF-α beingexpressed by T cells in B-CLL was positively correlated with tumour massand stage of the disease (Bojarska-Junak et al., 2002). Interleukin-1β(IL-1β) is a cytokine known to induce TNF-α production.

Thus, since the accumulation of excess cytokines and TNF-α can lead todeleterious consequences on the body, including cell death, there is aneed for a method to reduce the levels of cytokines in the body as wellas inhibiting or reducing apoptosis. The present invention fulfillsthese needs.

Deoxyhymisine synthase (DHS) and hypusine-containing eucaryotictranslation initiation Factor-5A (eIF-5A) are known to play importantroles in many cellular processes including cell growth anddifferentiation. Hypusine, a unique amino acid, is found in all examinedeucaryotes and archaebacteria, but not in eubacteria, and eIF-5A is theonly known hypusine-containing protein. Park (1988) J. Biol, Chem., 263,7447-7449; Schümann & Klink (1989) System: Appl. Microbiol., 11,103-107; Bartig et al. (1990) System. Appl. Microbiol., 13, 112-116;Gordon et al. (1987a) J. Biol., Chem., 262, 16585-16589. Active eIF-5Ais formed in two post-translational steps: the first step is theformation of a deoxyhypusine residue by the transfer of the 4-aminobutylmoiety of spermidine to the α-amino group of a specific lysine of theprecursor eIF-5A catalyzed by deoxyhypusine synthase; the second stepinvolves the hydroxylation of this 4-aminobutyl moiety by deoxyhypusinehydroxylase to form hypusine.

The amino acid sequence of eIF-5A is well conserved between species, andthere is strict conservation of the amino acid sequence surrounding thehypusine residue in eIF-5A, which suggests that this modification may beimportant for survival. Park et al. (1993) Biofactors, 4, 95-104. Thisassumption is further supported by the observation that inactivation ofboth isoforms of eIF-5A found to date in yeast, or inactivation of theDHS gene, which catalyzes the first step in their activation, blockscell division. Schnier et al. (1991) Mol. Cell. Biol., 11, 3105-3114;Sasaki et al. (1996) FEBS Lett., 384, 151-154; Park et al. (1998) J.Biol. Chem., 273, 1677-1683. However, depletion of eIF-5A protein inyeast resulted in only a small decrease in total protein synthesissuggesting that eIF-5A may be required for the translation of specificsubsets of mRNA's rather than for protein global synthesis. Kang et al.(1993), “Effect of initiation factor eIF-5A depletion on cellproliferation and protein synthesis,” in Tuite, M. (ed.), ProteinSynthesis and Targeting in Yeast, NATO Series H. The recent finding thatligands that bind eIF-5A share highly conserved motifs also supports theimportance of eIF-5A. Xu & Chen (2001) J. Biol. Chem., 276, 2555-2561.In addition, the hypusine residue of modified eIF-5A was found to beessential for sequence-specific binding to RNA, and binding did notprovide protection from ribonucleases.

In addition, intracellular depletion of eIF-5A results in a significantaccumulation of specific in RNAs in the nucleus, indicating that eIF-5Amay be responsible for shuttling specific classes of mRNAs from thenucleus to the cytoplasm. Liu & Tartakoff (1997) Supplement to MolecularBiology of the Cell, 8, 426a. Abstract No. 2476, 37th American Societyfor Cell Biology Annual Meeting. The accumulation of eIF-5A at nuclearpore-associated intranuclear filaments and its interaction with ageneral nuclear export receptor further suggest that eIF-5A is anucleocytoplasmic shuttle protein, rather than a component of polysomes.Rosorius et al. (1999) J. Cell Science, 112, 2369-2380.

The first cDNA for eIF-5A was cloned from human in 1989 by Smit-McBrideet al., and since then cDNAs or genes for eIF-5A have been cloned fromvarious eukaryotes including yeast, rat, chick embryo, alfalfa, andtomato. Smit-McBride et al. (1989) J. Biol. Chem., 264, 1578-1583;Schiller et al. (1991) (yeast); Sano, A. (1995) in Imahori, M. et al.(eds), Polyamines, Basic and Clinical Aspects, VNU Science Press, TheNetherlands, 81-88 (rat); Rinaudo & Park (1992) FASEB J., 6, A453 (chickembryo); Pay et al. (1991) Plant Mol. Biol., 17, 927-929 (alfalfa); Wanget al. (2001) J. Biol. Chem., 276, 17541-17549 (tomato).

Expression of eIF-5A mRNA has been explored in various human tissues andmammalian cell lines. For example, changes in eIF-5A expression havebeen observed in human fibroblast cells after addition of serumfollowing serum deprivation. Pang & Chen (1994) J. Cell Physiol., 160,531-538. Age-related decreases in deoxyhypusine synthase activity andabundance of precursor eIF-5A have also been observed in senescingfibroblast cells, although the possibility that this reflects averagingof differential changes in isoforms was not determined. Chen & Chen(1997) J. Cell Physiol., 170, 248-254.

Studies have shown that eIF-5A may be the cellular target of viralproteins such as the human immunodeficiency virus type 1 Rev protein andhuman T cell leukemia virus type 1 Rex protein. Ruhl et al. (1993) J.Cell Biol., 123, 1309-1320; Katahira et al. (1995) J. Virol., 69,3125-3133. Preliminary studies indicate that eIF-5A may target RNA byinteracting with other RNA-binding proteins such as Rev, suggesting thatthese viral proteins may recruit eIF-5A for viral RNA processing. Liu etal. (1997) Biol. Signals, 6, 166-174.

Thus, although eIF5A and DHS are known, there remains a need inunderstanding how these proteins are involved in apoptotic pathways aswell as cytokine stimulation to be able to modulate apoptosis andcytokine expression. The present invention fulfills this need.

SUMMARY OF INVENTION

The present invention relates to apoptosis specific eucaryoticinitiation factor 5A (eIF-5A), referred to as “apoptosis specificeIF-5A” or “eIF-5A1.” The present invention also relates toapoptosis-specific eIF-5A nucleic acids and polypeptides and methods forinhibiting or suppressing apoptosis in cells using antisense nucleotidesor siRNAs to inhibit expression of apoptosis-specific eIF-5A. Thepresent invention relates to a method of delivering siRNA to mammalianlung cells in vivo. The invention also relates to suppressing orinhibiting expression of pro-inflammatory cytokines by inhibitingexpression of apoptosis-specific eIF-5A. Further, the present inventionrelates to inhibiting or suppressing expression of p53 by inhibitingexpression of apoptosis-specific eIF-5A. The present invention alsorelates to a method of increasing Bcl-2 expression by inhibiting orsuppression expression of apoptosis factor 5A using antisensenucleotides or siRNAs. The present invention also provides a method ofinhibiting production of cytokines, especially TNF-α in human epithelialcells. In another embodiment of the present invention, suppressingexpression of apoptosis-specific eIF-5A by the use of antisenseoligonucleotides targeted at apoptosis-specific eIF-5A provides methodsof preventing retinal ganglion cell death in a glaucomatous eye.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence (SEQ ID NO: 11) and derived aminoacid sequence (SEQ ID NO: 12) of the 3’ end of rat apoptosis-specificeIF-5A.

FIG. 2 depicts the nucleotide sequence (SEQ ID NO: 15) and derived aminoacid sequence (SEQ ID NO: 16) of the 5 end of rat apoptosis-specificeIF-5A cDNA.

FIG. 3 depicts the nucleotide sequence of rat corpus luteumapoptosis-specific eIF-5A full-length cDNA (SEQ ID NO: 1). The aminoacid sequence is shown in SEQ ID NO: 2.

FIG. 4 depicts the nucleotide sequence (SEQ ID NO: 6) and derived aminoacid sequence (SEQ ID NO: 7) of the 3′ end of rat apoptosis-specific DHScDNA.

FIG. 5 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific eIF-5A cDNA (SEQ ID NO: 20) with thenucleotide sequence of human eIF-5A (SEQ ID NO: 3) (Accession numberBC000751 or NM_(—)001970, SEQ ID NO:3).

FIG. 6 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific eIF-5A cDNA (SEQ ID NO: 20) with thenucleotide sequence of human eIF-5A (SEQ NO: 4) (Accession numberNM-020390, SEQ ID NO:4).

FIG. 7 is an alignment of the full-length nucleotide sequence of ratcorpus luteum apoptosis-specific eIF-5A cDNA (SEQ ID NO: 20) with thenucleotide sequence of mouse eIF-5A (Accession number BC003889). Mousenucleotide sequence (Accession number BC003889) is SEQ ID NO:5.

FIG. 8 is an alignment of the derived full-length amino acid sequence ofrat corpus luteum apoptosis-specific eIF-5A (SEQ ID NO: 2) with thederived amino acid sequence of human eIF-5A (SEQ ID NO: 21) (Accessionnumber BC000751 or NM_(—)001970).

FIG. 9 is an alignment of the derived full-length amino acid sequence ofrat corpus luteum apoptosis-specific eIF-5A (SEQ ID NO: 2) with thederived amino acid sequence of human eIF-5A (SEQ ID NO: 22) (Accessionnumber NM_(—)20390).

FIG. 10 is an alignment of the derived full-length amino acid sequenceof rat corpus luteum apoptosis-specific eIF-5A. (SEQ ID NO: 2) with thederived amino acid sequence of mouse eIF-5A (SEQ ID NO: 23) (Accessionnumber BC003889).

FIG. 11 is an alignment of the partial-length nucleotide sequence of ratcorpus luteum apoptosis-specific DHS cDNA. (residues 1-453 of SEQ ID NO:6) with the nucleotide sequence of human DHS (SEQ ID NO: 8) (Accessionnumber BC000333, SEQ II) NO:8).

FIG. 12 is a restriction map of rat corpus luteum apoptosis-specificeIF-5A cDNA.

FIG. 13 is a restriction map of the partial-length ratapoptosis-specific DHS cDNA.

FIG. 14 is a Northern blot (top) and an ethidium bromide stained gel(bottom) of total RNA probed with the ³²P-dCTP-labeled 3′-end of ratcorpus luteum apoptosis-specific eIF-5A cDNA.

FIG. 15 is a Northern blot (top) and an ethidium bromide stained gel(bottom) of total RNA probed with the ³²P-dCTP-labeled 3′-end of ratcorpus luteum apoptosis-specific DHS cDNA.

FIG. 16 depicts a DNA laddering experiment in which the degree ofapoptosis in superovulated rat corpus lutea was examined after injectionwith PGF-2α.

FIG. 17 is an agarose gel of genomic DNA isolated from apoptosing ratcorpus luteum showing DNA laddering after treatment of rats with PGFF-2α.

FIG. 18 depicts a DNA laddering experiment in which the degree ofapoptosis in dispersed cells of superovulated rat corpora lutea wasexamined in rats treated with spermidine prior to exposure to PGF-2α.

FIG. 19 depicts a DNA laddering experiment in which the degree ofapoptosis in superovulated rat corpus lutea was examined in rats treatedwith spermidine and/or PGF-2α.

FIG. 20 is a Southern blot of rat genomic DNA probed with³²P-dCTP-labeled partial-length rat corpus luteum apoptosis-specificeIF-5A cDNA.

FIG. 21 depicts pHM6, a mammalian epitope tag expression vector (RocheMolecular Biochemicals).

FIG. 22 is a Northern blot (top) and ethidium bromide stained gel(bottom) of total RNA isolated from COS-7 cells after induction ofapoptosis by withdrawal of serum probed with the ³²P-dCTP-labeled3′-untranslated region of rat corpus luteum apoptosis-specific DHS cDNA.

FIG. 23 is a flow chart illustrating the procedure for transienttransfection of COS-7 cells.

FIG. 24 is a Western blot of transient expression of foreign proteins inCOS-7 cells following transfection with pHM6.

FIG. 25 illustrates enhanced apoptosis as reflected by increased caspaseactivity when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 26 illustrates enhanced apoptosis as reflected by increased DNAfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 27 illustrates detection of apoptosis as reflected by increasednuclear fragmentation when COS-7 cells were transiently transfected withpHM6 containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 28 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 29 illustrates detection of apoptosis as reflected byphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 30 illustrates enhanced apoptosis as reflected by increasedphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 31 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation.

FIG. 32 illustrates enhanced apoptosis when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation.

FIG. 33 illustrates down-regulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-specific eIF-5A in the sense orientation. The top photo is theCoomassie-blue-stained protein blot; the bottom photo is thecorresponding Western blot.

FIG. 34 is a Coomassie-blue-stained protein blot and the correspondingWestern blot of COS-7 cells transiently transfected with pHM6 containingfull-length rat apoptosis-specific eIF-5A in the antisense orientationusing Bcl-2 as a probe.

FIG. 35 is a Coomassie-blue-stained protein blot and the correspondingWestern blot of COS-7 cells transiently transfected with pHM6 containingfull-length rat apoptosis-specific eIF-5A in the sense orientation usingc-Myc as a probe.

FIG. 36 is a Coomassie-blue-stained protein blot and the correspondingWestern blot of COS-7 cells transiently transfected with pHM6 containingfull-length rat apoptosis-specific eIF-5A in the sense orientation whenp53 is used as a probe.

FIG. 37 is a Coomassie-blue-stained protein blot and the correspondingWestern blot of expression of pHM6-full-length rat apoptosis-specificeIF-5A in COS-7 cells using an anti-[HA]-peroxidase probe and aCoomassie-blue-stained protein blot and the corresponding Western blotof expression of pHM6-full-length rat apoptosis-specific eIF-5A in COS-7cells when a p53 probe is used.

FIG. 38 is an alignment of human eIF5A2 isolated from RKO cells (SEQ IDNO: 24) with the sequence of human eIF5A2 (SEQ D NO: 22) (Genbankaccession number XM_(—)113401). The consensus sequence is shown in SEQID NO: 28.

FIG. 39 is a graph depicting the percentage of apoptosis occurring inRKO and RKO-E6 cells following transient transfection. RKO and RKO-E6cells were transiently transfected with pHM6-LacZ orpHM6-apoptosis-specific eIF-5A. RKO cells treated with Actinomycin D andtransfected with pHM6-apoptosis-specific eIF-5A showed a 240% increasein apoptosis relative to cells transfected with pHM6-LacZ that were nottreated with Actinomycin D. RKO-E6 cells treated with Actinomycin D andtransfected with pHM6-apoptosis-specific eIF-5A showed a 105% increasein apoptosis relative to cells transfected with pHM6-LacZ that were nottreated with Actinomycin D.

FIG. 40 is a graph depicting the percentage of apoptosis occurring inRKO cells following transient transfection. RKO cells were transientlytransfected with pHM6-LacZ, pHM6-apoptosis-specific eIF-5A, pHM6-eIF5A2,or pHM6-truncated apoptosis-specific eIF-5A. Cells transfected withpHM6-apoptosis-specific eIF-5A showed a 25% increase in apoptosisrelative to control cells transfected with pHM6-LacZ. This increase wasnot apparent for cells transfected with pHM6-eIF5A2 or pHM6-truncatedapoptosis-specific eIF-5A.

FIG. 41 is a graph depicting the percentage of apoptosis occurring inRKO cells following transient transfection. RKO cells were either leftuntransfected or were transiently transfected with pHM6-LacZ orpHM6-apoptosis-specific eIF-5A. After correction for transfectionefficiency, 60% of the cells transfected with pHM6-apoptosis-specificeIF-5A were apoptotic.

FIG. 42 provides the results of a flow cytometry analysis of RKO cellapoptosis following transient transfection. RKO cells were either leftuntransfected or were transiently transfected with pHM6-LacZ,pHM6-apoptosis-specific eIF-5A, pHM6-eIF5A2, or pHM6-truncatedapoptosis-specific eIF-5A. The table depicts the percentage of cellsundergoing apoptosis calculated based on the area under the peak of eachgate. After correction for background apoptosis in untransfected cellsand for transfection efficiency, 80% of cells transfected withpHM6-apoptosis-specific eIF-5A exhibited apoptosis. Cells transfectedwith pHM6-LacZ, pHM6-eiF5A2 or pHM6-truncated apoptosis-specific eIF-5Aexhibited only background levels of apoptosis.

FIG. 43 provides Western blots of protein extracted from RKO cellstreated with 0.25 μg/ml Actinomycin D for 0, 3, 7, 24, and 48 hours. Thetop panel depicts a Western blot using anti-p53 as the primary antibody.The middle panel depicts a Western blot using anti-apoptosis-specificeIF-5A as the primary antibody. The bottom panel depicts the membraneused for the anti-apoptosis-specific eIF-5A blot stained with Coomassieblue following chemiluminescent detection to demonstrate equal loading.p53 and apoptosis-specific eIF-5A are both upregulated by treatment withActinomycin D.

FIG. 44 is a bar graph showing that both apoptosis-specific eIF-5A andproliferation eIF-5A are expressed in heart tissue. The heart tissue wastaken from patients receiving coronary artery bypass grafts (“CABG”).Gene expression levels apoptosis-specific eIF-5A (light gray bar) arecompared to proliferation eIF-5A (dark gray bar). The X-axis is patientidentifier numbers. The Y-axis is pg/ng of 18s (picograms of message RNAover nanograms of ribosomal RNA 18S).

FIG. 45 is a bar graph showing that both apoptosis-specific eIF-5A andproliferation eIF-5A are expressed in heart tissue. The heart tissue wastaken from patients receiving valve replacements. Gene expression levelsof apoptosis-specific eIF-5A (light gray bar) are compared toproliferation eIF-5A (dark gray bar). The X-axis is patient identifiernumbers. The Y-axis is pg/ng of 18s (picograms of message RNA overnanograms of ribosomal RNA 18S).

FIG. 46 is a bar graph showing the gene expression levels measured byreal-time PCR of apoptosis-specific eIF-5A (eIf5a) versus proliferationeIF-5A (eIF5b) in pre-ischemia heart tissue and post ischemia hearttissue. The Y-axis is pg/ng of 18s (picograms of message RNA overnanograms of ribosomal RNA 18S).

FIG. 47 depicts schematically an experiment performed on heart tissue.The heart tissue was exposed to normal oxygen levels and the expressionlevels apoptosis-specific eIF-5A and proliferating eIF-5A measured.Later, the amount of oxygen delivered to the heart tissue was lowered,thus inducing hypoxia and ischemia, and ultimately, a heart attack inthe heart tissue. The expression levels of apoptosis-specific eIF-5A andproliferating eIF-5A were measured and compared to the expression levelsof the heart tissue before it was damaged by ischemia.

FIG. 48 shows EKGs of heart tissue before and after the ischemia wasinduced.

FIG. 49 shows the lab bench with the set up of the experiment depictedin FIG. 47.

FIGS. 50A-F report patient data where the levels of apoptosis-specificeIF-5A are correlated with levels of IL-1β and IL-18. FIG. 50A is achart of data obtained from coronary artery bypass graft (CABG)patients. FIG. 50B is a chart of data obtained from valve replacementpatients. FIG. 50C is a graph depicting the correlation ofapoptosis-specific eIF-5A to IL-18 in CABG patients. FIG. 50D is a graphdepicting the correlation of proliferating eIF-5A to IL-18 in CABGpatients. FIG. 50E is a graph depicting the correlation ofapoptosis-specific eIF-5A to IL-18 in valve replacement patients. FIG.50F is a graph depicting the correlation of proliferating eIF-5A toIL-18 in valve replacement patients.

FIG. 51 is a chart of the patient's data from which patients data usedin FIGS. 50A-F was obtained.

FIG. 52 shows the levels of protein produced by RKO cells after beingtreated with antisense oligo 1, 2 and 3 (of apoptosis-specific eIF-5A)(SEQ ID NO: 35, 37 and 39, respectively). The RKO cells produced lessapoptosis-specific eIF-5A as well as less p53 after having beentransfected with the antisense apoptosis-specific eIF-5Aoligonucleotides.

FIG. 53 shows uptake of the fluorescently labeled antisenseoligonucleotide.

FIGS. 54-58 show a decrease in the percentage of cells undergoingapoptosis in the cells having being treated with antisenseapoptosis-specific eIF-5A oligonucleotides as compared to cells nothaving been transfected with the antisense apoptosis-specific eIF-5Aoligonucleotides.

FIG. 59 shows that treating lamina cribrosa cells with TNF-α and/orcamptothecin caused an increase in the number of cells undergoingapoptosis.

FIGS. 60 and 61 shows a decrease in the percentage of cells undergoingapoptosis in the cells having being treated with antisenseapoptosis-specific eIF-5A oligonucleotides as compared to cells nothaving been transfected with the antisense apoptosis-specific eIF-5Aoligonucleotides.

FIG. 62 shows that the lamina cribrosa cells uptake the labeled siRNA,either in the presence of serum or without serum.

FIG. 63 shows that cells transfected with apoptosis-specific eIF-5AsiRNA produced less apoptosis-specific eIF-5A protein and in addition,produced more Bcl-2 protein. A decrease in apoptosis-specific eIF-5Aexpression correlates with an increase in BCL-2 expression.

FIG. 64 shows that cells transfected with apoptosis-specific eIF-5AsiRNA produced less apoptosis factor 5a protein.

FIGS. 65-67 shows that cells transfected with apoptosis-specific eIF-5AsiRNA had a lower percentage of cells undergoing apoptosis afterexposure to amptothecin and TNF-α than untransfected cells.

FIG. 68 are photographs of Hoescht-stained lamina cribrosa cell line#506 transfected with siRNA and treated with camptothecin and TNF-α fromthe experiment described in FIG. 67 and Example 13. The apoptosing cellsare seen as more brightly stained cells. They have smaller nucleicbecause of chromatin condensation and are smaller and irregular inshape.

FIG. 69 shows that IL-1 exposed HepG2 cells transfected withapoptosis-specific eIF-5A cells secreted less TNF-α than non-transfectedcells.

FIG. 70 shows the sequence of human apoptosis-specific eIF-5A (SEQ IDNO:29) and the sequences of 5 siRNAs of the present invention (SEQ IDNO:30, 31, 32, 33 and 34).

FIG. 71 shows the sequence of human apoptosis-specific eIF-5A (SEQ IDNO: 29) and the sequences of 3 antisense oligonucleotides of the presentinvention (SEQ ID NO:35, 37, and 39, respectively in order ofappearance).

FIG. 72 shows the binding position of three antisense oligonucleotides(SEQ NO:25-27, respectively in order of appearance) targeted againsthuman apoptosis-specific eIF-5A. The full-length nucleotide sequence isSEQ ID NO: 19.

FIG. 73 a and b shows the nucleotide alignment (SEQ ID NO: 41 and 42,respectively in order of appearance) and amino acid alignment (SEQ IDNO: 43 and 22, respectively in order of appearance) of humanapoptosis-specific eIF-5A against human proliferating eIF-5A.

FIG. 74A provides a picture of a Western blot where siRNAs againstapoptosis-specific eIF-5A have reduced if not inhibited the productionof TNF-α in transfected HT-29 cells. FIG. 74B provides the results of anELISA.

FIG. 75 provides the results of an ELBA. TNF-α production was reduced incells treated with siRNAs against apoptosis-specific eIF-5A as comparedto control cells.

FIG. 76 shows the time course of the U-937 differentiation experiment.See Example 16.

FIG. 77 shows the results of a Western blot showing thatapoptosis-specific eIF-5A is up-regulated during monocytedifferentiation and subsequence TNF-α secretion.

FIG. 78 depicts stem cell differentiation and the use of siRNAs againstapoptosis-specific eIF-5A to inhibit cytokine production.

FIG. 79 is a bar graph showing that IL-8 is produced in response toTNF-alpha as well as in response to interferon. This graph shows thatsiRNA against apoptosis-specific eIF-5A blocked almost all IL-8 producedin response to interferon as well as a significant amount of the IL-8produced as a result of the combined treatment of interferon and TNF.

FIG. 80 is another bar graph showing that IL-8 is produced in responseto TNF-alpha as well as in response to interferon. This graph shows thatsiRNA against apoptosis-specific eIF-5A blocked almost all IL-8 producedin response to interferon as well as a significant amount of the IL-8produced as a result of the combined treatment of interferon and TNF.

FIG. 81 is a western blot of HT-29 cells treated with IFN gamma for 8and 24 hours. This blot shows up-regulation in HT-29 cells (4 fold at 8hours) of against apoptosis-specific eIF-5A in response to interferongamma.

FIG. 82 is a characterization of lamina cribrosa cells byimmunofluorescence. Lamina cribrosa cells (#506) isolated from the opticnerve head of an 83-year old male were characterized byimmunofluorescence. Primary antibodies were a) actin; b) fibronectin; c)laminin; d) GFAP. All pictures were taken at 400 times magnification.

FIG. 83 is a graph showing percent apoptosis of lamina cribrosa cellline #506 in response to treatment with camptothecin and TNF-α. Laminacribrosa cell line #506 cells were seeded at 40,000 cells per well ontoan 8-well culture slide. Three days later the confluent LC cells weretreated with either 10 ng/ml TNF-α, 50 LM camptothecin, or 10 ng/mlTNF-α plus 50 μM camptothecin. An equivalent volume of DMSO, a vehiclecontrol for camptothecin, was added to the untreated control cells. Thecells were stained with Hoescht 33258 48 hours after treatment andviewed by fluorescence microscopy using a UV filter. Cells with brightlystained condensed or fragmented nuclei were counted as apoptotic.

FIG. 84 shows expression levels of against apoptosis-specific eIF-5Aduring camptothecin or TNF-a plus camptothecin treatment. Laminacribrosa cell #506 cells were seeded at 40,000 cells per well onto a24-well plate. Three days later the LC cells were treated with either 50μM camptothecin or 10 ng/ml TNF-α plus 50 μM camptothecin and proteinlysate was harvested 1, 4, 8, and 24 hours later. An equivalent volumeof DMSO was Added to control cells as a vehicle control and cell lysatewas harvested 1 and 24 hours later. 5 μg of protein from each sample wasseparated by SDS-PAGE, transferred to a PVDF membrane, and Western blotwith anti-apoptosis-specific eIF-5A antibody. The bound antibody wasdetected by chemiluminescence and exposed to x-ray film. The membranewas then stripped and re-blotted with anti-β-actin as an internalloading control.

FIG. 85 shows expression levels of apoptosis-specific eIF-5A in laminacribosa cell lines #506 and #517 following transfection with siRNAs.Lamina cribrosa cell #506 and #517 cells were seeded at 10,000 cells perwell onto a 24-well plate. Three days later the LC cells weretransfected with either GAPDH siRNA, apoptosis-specific eIF-5A siRNAs#1-4 (SEQ ID NO:30-33) or control siRNA #5 (SEQ. ID NO:34). Three daysafter transfection the protein lysate was harvested and 5 μg of proteinfrom each sample was separated by SDS-PAGE, transferred to a PVDFmembrane, and Western blotted with anti-eIF5A antibody. The boundantibody was detected by chemiluminescence and exposed to x-ray film.The membrane was then stripped and re-blotted with anti-β-actin as aninternal loading control. This figure shows that cells treated withsiRNAs of apoptosis-specific eIF-5A produce less apoptosis-specificeIF-5A protein.

FIG. 86 shows the percent apoptosis of lamina cribosa cell line #506cells transfected with apoptosis-specific eIF-5A siRNAs and treated withTNF-α and camptothecin. Lamina cribrosa cell line #506 cells were seededat 7500 cells per well onto an 8-well culture slide. Three days laterthe LC cells were transfected with either GAPDH siRNA,apoptosis-specific eIF-5A siRNAs #1-4 (SEQ ID NO:30-33), or controlsiRNA #5 (SEQ ID NO:34). 72 hours after transfection, the transfectedcells were treated with 10 ng/ml TNF-α plus 50 μM camptothecin.Twenty-four hours later the cells were stained with Hoescht 33258 andviewed by fluorescence microscopy using a UV filter. Cells with brightlystained condensed or fragmented nuclei were counted as apoptotic. Thisgraph represents the average of n=4 independent experiments. This figureshows that cells treated with siRNAs of apoptosis-specific eIF-5A show asmaller percentage of apoptosis upon treatment with camptothecin and TNFas compared to cells not transfected with siRNAs of apoptosis-specificeIF-5A.

FIG. 87 shows percent apoptosis of lamina cribosa cell line #517 cellstransfected with apoptosis-specific eIF-5A siRNA #1 and treated withTNF-α and camptothecin. Lamina cribrosa cell line #517 cells were seededat 7500 cells per well onto an 8-well culture slide. Three days laterthe LC cells were transfected with either apoptosis-specific eIF-5AsiRNA #1 (SEQ ID NO:30) or control siRNA #5 (SEQ ID NO:34). 72 hoursafter transfection, the transfected cells were treated with 10 ng/mlTNF-α plus 50 μM camptothecin. Twenty-four hours later the cells werestained with Hoescht 33258 and viewed by fluorescence microscopy using aUV filter. Cells with brightly stained condensed or fragmented nucleiwere counted as apoptotic. The results of two independent experimentsare represented here. This shows that cells treated with siRNAs had alower percentage of apoptosis.

FIG. 88 shows TUNEL-labeling of lamina cribosa cell line #506 cellstransfected with apoptosis-specific eIF-5A siRNA #1 and treated withTNF-α and camptothecin. Lamina cribrosa cell line #506 cells were seededat 7500 cells per well onto an 8-well culture slide. Three days laterthe LC cells were transfected with either apoptosis-specific eIF-5AsiRNA #1 (SEQ ID NO:30) or control siRNA #5 (SEQ ID NO:34). 72 hoursafter transfection, the transfected cells were treated with 10 ng/mlTNF-α plus 50 μM camptothecin. Twenty-four hours later the cells werestained with Hoescht 33258 and DNA fragmentation was evaluated in situusing the terminal deoxynucleotidyl transferase-mediateddUTP-digoxigenin nick end labeling (TUNEL) method. Panel A representsthe slide observed by fluorescence microscopy using a fluorescein filterto visualize TUNEL-labeling of the fragmented DNA of apoptotic cells.Panel B represents the same slide observed by through a UV filter tovisualize the Hoescht-stained nuclei. The results are representative oftwo independent experiments. All pictures were taken at 400 timesmagnification.

FIG. 89 depicts the design of siRNAs against apoptosis-specific eIF-5A.The siRNAs have the SEQ ID NO: 45, 48, 51, 54 and 56. The full-lengthnucleotide sequence is shown in SEQ ID NO: 29.

FIG. 90 shows the results of an experiment where siRNAs directed againstapoptosis-specific eIF-5A provided for a reduction in NKkB activation inthe presence of interferon gamma and LPS.

FIG. 91 shows the time course for PBMC experiments (see Example 18).

FIG. 92 shows a Western blot of a cell lysate from PBMCs collected fromtwo donors over a time course. The PBMCs were treated with PMA andsubsequently stimulated with LPS to have an increased apoptosis-specificeIF-5A expression.

FIG. 93 shows that PBMCs treated with PMA and subsequently stimulatedwith LPS have an increased apoptosis-specific eIF-5A expression, whichcoincides with increased TNF production.

FIG. 94 demonstrates that PBMCs respond to LPS without PMAdifferentiation.

FIG. 95 shows that PBMCs transfected with apoptosis-specific eIF-5AsiRNAs demonstrate suppression of expression of apoptosis-specificeIF-5A.

FIG. 96 shows that PBMCs transfected with apoptosis-specific eIF-5AsiRNAs and stimulated with LPS produce less TNF than PBMCs nottransfected with apoptosis-specific eIF-5A siRNAs.

FIG. 97 shows a western blot of cell lysate from HT-29 cells treatedwith or without gamma interferon.

FIG. 98 shows a western blot of cell lysate from HT-29 cells that weretransfected with either control siRNA or apoptosis-specific eIF-5AsiRNAs. This figure shows that siRNAs of apoptosis-specific eIF-5Ainhibit expression of apoptosis-specific eIF-5A.

FIG. 99 shows that HT-29 cells transfected with apoptosis-specificeIF-5A siRNAs have a reduced level of TNF production.

FIG. 100 shows that HT-29 cells transfected with apoptosis-specificeIF-5A siRNAs exhibit a decreased in apoptosis as compared to controlcells. Both control and siRNA-transfected cells were primed withinterferon gamma and also treated with TNF-α.

FIG. 101 shows that HT-29 cells transfected with apoptosis-specificeIF-5A siRNAs express less TLR4 protein than control cells.

FIG. 102 shows that HT-29 cells transfected with apoptosis-specificeIF-5A siRNAs express less TNFR1 protein than control cells.

FIG. 103 shows that HT-29 cells transfected with apoptosis-specificeIF-5A siRNAs express less iNOS protein than control cells.

FIG. 104 shows that HT-29 cells transfected with apoptosis-specificeIF-5A siRNAs express less TLR4 mRNA than control cells.

FIG. 105 shows the time course for U937 treatments.

FIG. 106 shows that apoptosis-specific eIF-5A is upregulated with PMA inU937 cells.

FIG. 107 shows that apoptosis-specific eIF-5A is upregulated with LPS inU937 cells.

FIG. 108 shows that apoptosis-specific eIF-5A protein expression isstill reduced after numerous hours following siRNA treatment.

FIG. 109 shows that siRNA mediated down-regulation of apoptosis-specificeIF-5A coincides with a reduction of TLR4.

FIG. 110 shows that siRNA mediated down-regulation of apoptosis-specificeIF-5A coincides with fewer glycosylated forms of the interferon gammareceptor in U937 cells.

FIG. 111 shows that siRNA mediated down-regulation of apoptosis-specificeIF-5A coincides with a reduction in TNFR1 in U937 cells.

FIG. 112 shows that siRNA mediated down-regulation of apoptosis-specificeIF-5A coincides with a reduction in LPS-induced TNF-α production inU937 cells.

FIG. 113 shows that siRNA mediated down-regulation of apoptosis-specificeIF-5A coincides with a reduction in LPS-induced IL-1β production inU937 cells.

FIG. 114 shows that siRNA mediated down-regulation of apoptosis-specificeIF-5A coincides with a reduction in LPS-induced IL-8 production in U937cells.

FIG. 115 shows that IL-6 production is independent of siRNA mediateddown-regulation of apoptosis-specific eIF-5A in U937 cells.

DETAILED DESCRIPTION OF THE INVENTION

Several isoforms of eukaryotic initiation factor 5A (“eIF-5A”) have beenisolated and present in published databanks. It was thought that theseisoforms were functionally redundant. The present inventors havediscovered that one isoform is upregulated immediately before theinduction of apoptosis, which they have designated apoptosis-specificeIF-5A or eIF-5A1. The subject of the present invention isapoptosis-specific eIF-5A and DHS, which is involved in the activationof eIF-5A.

Apoptosis-specific eIF-5A is likely to be a suitable target forintervention in apoptosis-causing disease states since it appears to actat the level of post-transcriptional regulation of downstream effectorsand transcription factors involved in the apoptotic pathway.Specifically, apoptosis-specific eIF-5A appears to selectivelyfacilitate the translocation of mRNAs encoding downstream effectors andtranscription factors of apoptosis from the nucleus to the cytoplasm,where they are subsequently translated. The ultimate decision toinitiate apoptosis appears to stern from a complex interaction betweeninternal and external pro- and anti-apoptotic signals. Lowe & Lin (2000)Carcinogenesis, 21, 485-495. Through its ability to facilitate thetranslation of downstream apoptosis effectors and transcription factors,apoptosis-specific eIF-5A appears to tip the balance between thesesignals in favor of apoptosis.

Accordingly, the present invention provides a method of suppressing orreducing apoptosis in a cell by administering an agent that inhibits orreduces expression of either apoptosis-specific eIF-5A or DHS. Byreducing expression of DHS, there is less DHS protein to be available toactivate apoptosis-specific eIF-5A. One agent that can inhibit or reduceexpression of apoptosis-specific eIF-5A or DHS are antisenseoligonucleotides of apoptosis-specific eIF-5A or DHS. By reducingactivation of apoptosis-specific eIF-5A or by reducing or inhibitingexpression of apoptosis-specific eIF-5A, cellular apoptosis can bedelayed or inhibited.

Antisense oligonucleotides have been successfully used to accomplishboth in vitro as well as in vivo gene-specific suppression. Antisenseoligonucleotides are short, synthetic strands of DNA (or DNA analogs),RNA (or RNA analogs), or DNA/RNA hybrids that are antisense (orcomplimentary) to a specific DNA or RNA target. Antisenseoligonucleotides are designed to block expression of the protein encodedby the DNA or RNA target by binding to the target mRNA and haltingexpression at the level of transcription, translation, or splicing. Byusing modified backbones that resist degradation (Blake et al., 1985),such as replacement of the phosphodiester bonds in the oligonucleotideswith phosphorothioate linkages to retard nuclease degradation (Matzuraand Eckstein, 1968), antisense oligonucleotides have been usedsuccessfully both in cell cultures and animal models of disease(Hogrefe, 1999). Other modifications to the antisense oligonucleotide torender the oligonucleotide more stable and resistant to degradation areknown and understand by one skilled in the art. Antisenseoligonucleotide as used herein encompasses double stranded or singlestranded DNA, double stranded or single stranded RNA, DNA/RNA hybrids,DNA and RNA analogs, and oligonucleotides having base, sugar, orbackbone modifications. The oligonucleotides may be modified by methodsknown in the art to increase stability, increase resistance to nucleasedegradation or the like. These modifications are known in the art andinclude, but are not limited to modifying the backbone of theoligonucleotide, modifying the sugar moieties, or modifying the base.

Preferably, the antisense oligonucleotides of the present invention havea nucleotide sequence encoding a portion or the entire coding sequenceof an apoptosis-specific eIF-5A polypeptide or a DHS polypeptide. Theinventors have transfected various cell lines with antisense nucleotidesencoding a portion of an apoptosis-specific eIF-5A polypeptide asdescribed below and measured the number of cells undergoing apoptosis.The cell populations that were transfected with the antisenseoligonucleotides showed a decrease in the number of cells undergoingapoptosis as compared to like cell populations not having beentransfected with the antisense oligos. FIGS. 54-58 show a decrease inthe percentage of cells undergoing apoptosis in the cells having beingtreated with antisense apoptosis-specific eIF-5A oligonucleotides ascompared to cells not having been transfected with the antisenseapoptosis-specific eIF-5A oligonucleotides.

The present invention contemplates the use of many suitable nucleic acidsequences encoding an apoptosis-specific eIF-5A polypeptide or DHSpolypeptide. For example, the present invention provides antisenseoligonucleotides of the following apoptosis-specific eIF-5A nucleic acidsequences (SEQ ID NOS:1, 3, 4, 5, 11, 12, 15, 16, 19, 20, and 21) andDHS sequences (SEQ ID NOS:6, 7, 8). Antisense oligonucleotides of thepresent invention need not be the entire length of the provided SEQ IDNOs. They need only be long enough to be able to bind to the mRNA andinhibit expression of such mRNA. Inhibition or reduction of expression”or “suppression of expression” refers to the absence or detectabledecrease in the level of protein and/or mRNA product from a target gene,such as apoptosis-specific eIF-5A.

Exemplary antisense oligonucleotides of apoptosis-specific eIF-5A thatdo not comprise the entire coding sequence are antisenseoligonucleotides of apoptosis-specific eIF-5A having the following SEQID NO: 35, 37, and 39.

“Antisense oligonucleotide of apoptosis-specific eIF-5A” includesoligonucleotides having substantial sequence identity or substantialhomology to apoptosis-specific eIF-5A. Additional antisenseoligonucleotides of apoptosis-specific eIF-5A of the present inventioninclude those that have substantial sequence identity to thoseenumerated above (i.e. 90% homology) or those having sequences thathybridize under highly stringent conditions to the enumerated SEQ EDNOs. As used herein, the term “substantial sequence identity” or“substantial homology” is used to indicate that a sequence exhibitssubstantial structural or functional equivalence with another sequence.Any structural or functional differences between sequences havingsubstantial sequence identity or substantial homology will be deminimus; that is, they will not affect the ability of the sequence tofunction as indicated in the desired application. Differences may be dueto inherent variations in codon usage among different species, forexample. Structural differences are considered de minimus if there is asignificant amount of sequence overlap or similarity between two or moredifferent sequences or if the different sequences exhibit similarphysical characteristics even if the sequences differ in length orstructure. Such characteristics include, for example, the ability tohybridize under defined conditions, or in the case of proteins,immunological crossreactivity, similar enzymatic activity, etc. Theskilled practitioner can readily determine each of these characteristicsby art known methods.

Additionally, two nucleotide sequences are “substantially complementary”if the sequences have at least about 70 percent or greater, morepreferably 80 percent or greater, even more preferably about 90 percentor greater, and most preferably about 95 percent or greater sequencesimilarity between them. Two amino acid sequences are substantiallyhomologous if they have at least 70%, more preferably at least 80%, evenmore preferably at least 90%, and most preferably at least 95%similarity between the active, or functionally relevant, portions of thepolypeptides.

To determine the percent identity of two sequences, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregardedfor comparison purposes). In a preferred embodiment, at least 30%, 40%,50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequenceis aligned for comparison purposes. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991).

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram. BLAST protein searches can be performed with the XBLAST programto obtain amino acid sequences homologous to the proteins of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al. (1997) NucleicAcids Res. 25(17):3389-3402. When utilizing BLAST and gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

The term “apoptosis-specific eIF-5A° ′ includes functional derivativesthereof. The term “functional derivative” of a nucleic acid is usedherein to mean a homolog or analog of the gene or nucleotide sequence. Afunctional derivative may retain the function of the given gene, whichpermits its utility in accordance with the invention. “Functionalderivatives” of the apoptosis-specific eIF-5A polypeptide or functionalderivatives of antisense oligonucleotides of apoptosis-specific eIF-5Aas described herein are fragments, variants, analogs, or chemicalderivatives of apoptosis-specific eIF-5A that retain Apoptosis-specificeIF-5A activity or immunological cross reactivity with an antibodyspecific for apoptosis-specific eIF-5A. A fragment of theapoptosis-specific eIF-5A polypeptide refers to any subset of themolecule.

Functional variants can also contain substitutions of similar aminoacids that result in no change or an insignificant change in function.Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al. (1989) Science 244:1081-1085). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as kinase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al. (1992) J. Mol. Biol. 224:899-904; de Vos et al.(1992) Science 255:306-312).

A “variant” refers to a molecule substantially similar to either theentire gene or a fragment thereof, such as a nucleotide substitutionvariant having one or more substituted nucleotides, but which maintainsthe ability to hybridize with the particular gene or to encode mRNAtranscript which hybridizes with the native DNA. A “homolog” refers to afragment or variant sequence from a different animal genus or species.An “analog” refers to a non-natural molecule substantially similar to orfunctioning in relation to the entire molecule, a variant or a fragmentthereof.

Variant peptides include naturally occurring variants as well as thosemanufactured by methods well known in the art. Such variants can readilybe identified/made using molecular techniques and the sequenceinformation disclosed herein. Further, such variants can readily bedistinguished from other proteins based on sequence and/or structuralhomology to the eIF-5A or DHS proteins of the present invention. Thedegree of homology/identity present will be based primarily on whetherthe protein is a functional variant or non-functional variant, theamount of divergence present in the paralog family and the evolutionarydistance between the orthologs.

Non-naturally occurring variants of the eIF-5A or DHS polynucleotides,antisense oligonucleotides, or proteins of the present invention canreadily be generated using recombinant techniques. Such variantsinclude, but are not limited to deletions, additions and substitutionsin the nucleotide or amino acid sequence. For example, one class ofsubstitutions are conserved amino acid substitutions. Such substitutionsare those that substitute a given amino acid in a protein by anotheramino acid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gin; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

The term “hybridization” as used herein is generally used to meanhybridization of nucleic acids at appropriate conditions of stringencyas would be readily evident to those skilled in the art depending uponthe nature of the probe sequence and target sequences. Conditions ofhybridization and washing are well known in the art, and the adjustmentof conditions depending upon the desired stringency by varyingincubation time, temperature and/or ionic strength of the solution arereadily accomplished. See, e.g. Sambrook, J. et. al., Molecular Cloning:A Laboratory Manual, 2^(nd) edition, Cold Spring Harbour Press, ColdSpring Harbor, N.Y., 1989.

The choice of conditions is dictated by the length of the sequencesbeing hybridized, in particular, the length of the probe sequence, therelative G-C content of the nucleic acids and the amount of mismatchesto be permitted. Low stringency conditions are preferred when partialhybridization between strands that have lesser degrees ofcomplementarity is desired. When perfect or near perfect complementarityis desired, high stringency conditions are preferred. High stringencyconditions means that the hybridization solution contains 6×S.S.C., 0.01M EDTA, 1×Denhardt's solution and 0.5% SDS. Hybridization is carried outat about 68° C. for about 3 to 4 hours for fragments of cloned DNA andfor about 12 to 16 hours for total eucaryotic DNA. For lowerstringencies, the temperature of hybridization is reduced to about 42°C. below the melting temperature (T_(m)) of the duplex. The T_(m) isknown to be a function of the G-C content and duplex length as well asthe ionic strength of the solution.

As used herein, the phrase “hybridizes to a corresponding portion” of aDNA or RNA molecule means that the molecule that hybridizes, e.g.,oligonucleotide, polynucleotide, or any nucleotide sequence (in sense orantisense orientation) recognizes and hybridizes to a sequence inanother nucleic acid molecule that is of approximately the same size andhas enough sequence similarity thereto to effect hybridization underappropriate conditions. For example, a 100 nucleotide long sensemolecule will recognize and hybridize to an approximately 100 nucleotideportion of a nucleotide sequence, so long as there is about 70% or moresequence similarity between the two sequences. It is to be understoodthat the size of the “corresponding portion” will allow for somemismatches in hybridization such that the “corresponding portion” may besmaller or larger than the molecule which hybridizes to it, for example20-30% larger or smaller, preferably no more than about 12-15% larger orsmaller.

In addition, functional variants of polypeptides can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Amino acids that are essential forfunction can be identified by methods known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunningham etal., Science 244:1081-1085 (1989)). The latter procedure introducessingle alanine mutations at every residue in the molecule. The resultingmutant molecules are then tested for biological activity or in assays.

The present invention also provides other agents that can inhibit orreduce expression of apoptosis-specific eIF-5A or DHS. One such agentincludes small inhibitory RNAs (“siRNA”). siRNA technology has beenemerging as a viable alternative to antisense oligonucleotides sincelower concentrations are required to achieve levels of suppression thatare equivalent or superior to those achieved with antisenseoligonucleotides (Thompson, 2002). Long double-stranded RNAs have beenused to silence the expression of specific genes in a variety oforganisms such as plants, nematodes, and fruit flies. An RNase-IIIfamily enzyme called Dicer processes these long double stranded RNAsinto 21-23 nucleotide small interfering RNAs which are then incorporatedinto an RNA-induced silencing complex (RISC). Unwinding of the siRNAactivates RISC and allows the single-stranded siRNA to guide the complexto the endogenous mRNA by base pairing. Recognition of the endogenousmRNA by RISC results in its cleavage and consequently makes itunavailable for translation. Introduction of long double stranded RNAinto mammalian cells results in a potent antiviral response, which canbe bypassed by use of siRNAs. (Elbashir et al., 2001). siRNA has beenwidely used in cell cultures and routinely achieves a reduction inspecific gene expression of 90% or more.

The use of siRNAs has also been gaining popularity in inhibiting geneexpression in animal models of disease. A recent study demonstrated thatan siRNA against luciferase was able to block luciferase expression froma co-transfected plasmid in a wide variety of organs in post-natal mice.(Lewis et al., 2002). An siRNA against Fas, a receptor in the TNFfamily, injected hydrodynamically into the tail vein of mice was able totransfect greater than 80% of hepatocytes and decrease Fas expression inthe liver by 90% for up to 10 days after the last injection (Song etal., 2003). The Fas siRNA was also able to protect mice from liverfibrosis and fulminant hepatitis. The development of sepsis in micetreated with a lethal dose of lipopolysaccharide was inhibited by theuse of an siRNA directed against TNF-α (Sørensen et al., 2003). SiRNAhas the potential to be a very potent drug for the inhibition ofspecific gene expression in vitro in light of their long-lastingeffectiveness in cell cultures and their ability to transfect cells invivo and their resistance to degradation in serum in vivo (Bertrand etal., 2002) in vivo.

The present inventors have transfected cells with siRNAs ofapoptosis-specific eIF-5A and studied the effects on expression ofapoptosis-specific eIF-5A. FIG. 64 shows that cells transfected withapoptosis-specific eIF-5A siRNA produced less apoptosis-specific eIF-5Aprotein. FIGS. 65-67 show that cell populations transfected withapoptosis-specific eIF-5A siRNAs have a lower percentage of cellsundergoing apoptosis after exposure to amptothecin and TNF-α as comparedto cells not having been transfected with apoptosis-specific eIF-5AsiRNAs. Thus, one embodiment of the present invention provides forinhibiting expression of apoptosis-specific eIF-5A in cells bytransfecting the cells with a vector comprising a siRNAs ofapoptosis-specific eIF-5A.

Preferred siRNAs of apoptosis-specific eIF-5A include those that haveSEQ ID NO: 31, 31, 32, and 33. Additional siRNAs include those that havesubstantial sequence identity to those enumerated (i.e. 90% homology) orthose having sequences that hybridize under highly stringent conditionsto the enumerated SEQ NOs. What is meant by substantial sequenceidentity and homology is described above with respect to antisenseoligonucleotides of the present invention. The term “siRNAs ofapoptosis-specific eIF-5A” include functional variants or derivatives asdescribed above with respect to antisense oligonucleotides of thepresent invention.

Delivery of siRNA and expression constructs/vectors comprising siRNA areknown by those skilled in the art. U.S. applications 2004/106567 and2004/0086884, which are herein incorporated by reference in theirentirety, provide numerous expression constructs/vectors as well asdelivery mechanism including viral vectors, non viral vectors, liposomaldelivery vehicles, plasmid injection systems, artificial viral envelopesand poly-lysine conjugates to name a few.

One skilled in the art would understand regulatory sequences useful inexpression constructs/vectors with antisense oligonucleotides or siRNA.For example, regulatory sequences may be a constitutive promoter, aninducible promoter, a tissue-specific promoter, or a combinationthereof.

Many important human diseases are caused by abnormalities in the controlof apoptosis. These abnormalities can result in either a pathologicalincrease in cell number (e.g. cancer) or a damaging loss of cells (e.g.degenerative diseases). As non-limiting examples, the methods andcompositions of the present invention can be used to prevent or treatthe following apoptosis-associated diseases and disorders:neurological/neurodegenerative disorders (e.g., Alzheimer's,Parkinson's, Huntington's, Amyotrophic Lateral Sclerosis (Lou Gehrig'sDisease), autoimmune disorders (e.g., rheumatoid arthritis, systemiclupus erythematosus (SLE), multiple sclerosis), Duchenne MuscularDystrophy (DMD), motor neuron disorders, ischemia, heart ischemia,chronic heart failure, stroke, infantile spinal muscular atrophy,cardiac arrest, renal failure, atopic dermatitis, sepsis and septicshock; AIDS, hepatitis, glaucoma, diabetes (type 1 and type 2), asthma,retinitis pigmentosa, osteoporosis, xenograft rejection, and burninjury.

One such disease caused by abnormalities in the control of apoptosis isglaucoma. Apoptosis in various optical tissues is a criticalfactor-leading to blindness in glaucoma patients. Glaucoma is a group ofeye conditions arising from damage to the optic nerve that results inprogressive blindness. Apoptosis has been shown to be a direct cause ofthis optic nerve damage.

Early work in the field of glaucoma research has indicated that elevatedintra-ocular pressure (“IOP”) leads to interference in axonal transportat the level of the lamina cribosa (a perforated, collagenous connectivetissue) that is followed by the death of retinal ganglion cells. Quigleyand Anderson (1976) Invest. Ophthalmol, Vis. Sci., 15, 606-16; Minckler.Bunt, and Klock, (1978) Invest. Ophthalmol. Vis. Sci., 17, 33-50;Anderson and Hendrickson, (1974) Invest. Ophthalmol. Vis, Sci., 13,771-83; Quigley et al., (1980) Invest. Ophthalmol. Vis. Sci., 19,505-17. Studies of animal models of glaucoma and post-mortem humantissues indicate that the death of retinal ganglion cells in glaucomaoccurs by apoptosis. Garcia-Valenzuela et. al., (1995) Exp. Eye Res.,61, 33-44; Quigley et al., (1995) Invest. Ophthalmol. Vis. Sci., 36,774-786; Monard, (1998) In: Haefliger I O, Flammer J (eds) Nitric oxideand Endothelin in the Pathogenesis of Glaucoma, New York, N.Y.,Lippincott-Raven, 213-220. The interruption of axonal transport as aresult of increased LOP may contribute to retinal ganglion cell death bydeprivation of trophic factors. Quigley, (1995) Aust NZJ Ophthalmol,23(2), 85-91. Optic nerve head astrocytes in glaucomatous eyes have alsobeen found to produce increased levels of some neurotoxic substances.For example, increased production of tumor necrosis factor-α (TNF-α)(Yan et al., (2000) Arch. Ophthalmol., 118, 666-673), and nitric oxidesynthase (Neufeld et al., (1997) Arch. Ophthalmol., 115, 497-503), theenzyme which gives rise to nitric oxide, has been found in the opticnerve head of glaucomatous eyes. Furthermore, increased expression ofthe inducible form of nitric oxide synthase (iNOS) and TNF-α byactivated retinal glial cells have been observed in rat models ofhereditary retinal diseases. Cotinet et al., (1997) Glia, 20, 59-69; deKozak et al., (1997) Ocul. Immunoi. Inflamm., 5, 85-94; Goureau et al.,(1999) J. Neurochem, 72, 2506-2515. In the glaucomatous optic nervehead, excessive nitric oxide has been linked to“the degeneration ofaxons of retinal ganglion cells. Arthur and Neufeld, (1999) SurvOphthalmol, 43 (Suppl 1), S129-S135. Finally, increased production ofTNF-α by retinal glial cells in response to simulated ischemia orelevated hydrostatic pressure has been shown to induce apoptosis inco-cultured retinal ganglion cells. Tezel and Wax, (2000) J. Neurosci.,20(23), 8693-8700.

Protecting retinal ganglion cells from degeneration by apoptosis isunder study as a potential new treatment for blindness due to glaucoma.Antisense oligonucleotides have been used successfully in animal modelsof eye disease. In a model of transient global retinal ischemia,expression of caspase 2 was increased during ischemia, primarily in theinner nuclear and ganglion cell layers of the retina. Suppression ofcaspase using an antisense oligonucleotide led to significanthistopathologic and functional improvement as determined byelectroretinogram. Singh et al., (2001) J. Neurochem., 77(2), 466-75.Another study demonstrated that, upon transfection of the optic nerve,retinal ganglion cells upregulate the pro-apoptotic protein Bax andundergo apoptosis. Repeated injections of a Bax antisenseoligonucleotide into the temporal superior retina of rats inhibited thelocal expression of Bax and increased the number of surviving retinalganglion cells following transaction of the optic nerve. Isenmann etal., (1999) Cell Death Differ., 6(7). 673-82.

Delivery of antisense oligonucleotides to retinal ganglion cells hasbeen improved by encapsulating the oligonucleotides in liposomes, whichwere then coated with the envelope of inactivated hemagglutinating virusof Japan (HVJ; Sendai virus) by fusion (HVJ liposomes). Intravitrealinjection into mice of FITC-labeled antisense oligonucleotidesencapsulated in HVJ liposomes resulted in high fluorescence within 44%of the cells in the ganglion layer which lasted three days whilefluorescence with naked FITC-labeled antisense oligonucleotidedisappeared after one day. Hangai et al., (1998) Arch Ophthalmol,116(7), 976.

One method of preventing or reducing apoptosis of the present inventionis directed to preventing or reducing apoptosis in cells and tissues ofthe eye, such as but not limited to, astrocytes, retinal ganglion.,retinal glial cells and lamina cribosa. Death of retinal ganglion cellsin glaucoma occurs by apoptosis and which leads to blindness. Thus,providing a method of inhibiting or reducing apoptosis in retinalganglion cells or by protecting retinal ganglion cells from degenerationby apoptosis provides a novel treatment for prevention of blindness dueto glaucoma.

The present invention provides a method for preventing or inhibitingretinal ganglion cell death in a glaucomatous eye, by suppressingexpression of apoptosis-specific eIF-5A. Inhibiting the expression ofapoptosis-specific eIF-5A reduces apoptosis. Apoptosis-specific eIF-5Ais a powerful gene that appears to regulate the entire apoptoticprocess. Thus, controlling apoptosis in the optic nerve head indicatesthat blocking expression of apoptosis-specific eIF-5A provides atreatment for glaucoma.

Suppression of expression of apoptosis-specific eIF-5A is accomplishedby administering an antisense oligonucleotides or a siRNA of humanapoptosis-specific eIF-5A to cells of the eye such as, but not limitedto lamina cribrosa, astrocytes, retinal ganglion, or retinal glialcells. Antisense oligonucleotides and siRNAs are as defined above, i.e.have a nucleotide sequence encoding at least a portion of anapoptosis-specific eIF-5A polypeptide. Exemplary antisenseoligonucleotides useful in this aspect of the invention comprise SEQ IDNO:26 or 27 or oligonucleotides that bind to a sequence complementary toSEQ ID NO:26 or 27 under high stringency conditions and which inhibitexpression of apoptosis-specific eIF-5A.

Another embodiment of the invention provides a method of suppressingexpression of apoptosis-specific eIF-5A in lamina cribosa cells,astrocyte cells, retinal ganglion cells or retinal glial cells.Antisense oligonucleotides or siRNAs, such as but not limited to, SEQ IDNO:26 and 27, targeted against human apoptosis-specific eIF-5A areadministered to lamina cribosa cells, astrocyte cells, retinal ganglioncells or retinal glial cells. The cells may be of human origin.

In addition to having a role in apoptosis, eIF5A may also play a role inthe immune response. The present inventors have discovered thatapoptosis-specific eIF-5A levels correlate with elevated levels of twocytokines (Interleukin 1-beta “IL-1β” and interleukin 18 “IL-18”) inischemic heart tissue, thus further proving that apoptosis-specificeIF-5A is involved in cell death as it is present in ischemic hearttissue. This apoptosis-specific eIF-5A/interleukin correlation is notseen in non-ischemic heart tissue. See FIGS. 50A-F and 51. Using PCRmeasurements, levels of apoptosis-specific eIF-5A, and proliferatingeIF-5A (“eIF-5A2”)—another isoform), IL-1β, and IL-18 were measured andcompared in various ischemic heart tissue (from coronary bypass graftand valve (mitral and atrial valve) replacement patients).

The correlation of apoptosis-specific eIF-5A to these potentinterleukins further suggests that the inflammation and apoptosispathways in ischemia may be controlled via controlling levels ofapoptosis-specific eIF-5A. Further evidence that apoptosis-specificeIF-5A is involved in the immune response is suggested by the fact thathuman peripheral blood mononuclear cells (PBMCs) normally express verylow levels of eIF-5A, but upon stimulation with T-lymphocyte-specificstimuli expression of apoptosis-specific eIF-5A increases dramatically(Bevec et al., 1994). This suggests a role for apoptosis-specific eIF-5Ain T-cell proliferation and/or activation. Since activated T cells arecapable of producing a wide variety of cytokines; it is also possiblethat apoptosis-specific eIF-5A may be required as a nucleocytoplasmicshuttle for cytokine mRNAs. The authors of the above referenced articlealso found elevated levels of eIF5A in the PBMCs of HIV-1 patients whichmay contribute to efficient HIV replication in these cells as eIF5A hasbeen demonstrated to be a cellular binding factor for the HIV Revprotein and required for HIV replication (Ruhl et al., 1993).

More recently, eIF-5A expression was found to be elevated duringdendritic cell maturation (Kruse et al., 2000). Dendritic cells areantigen-presenting cells that sensitize helper and killer T cells toinduce T cell-mediated immunity (Steinman, 1991). Immature dendriticcells lack the ability to stimulate T cells and require appropriatestimuli (i.e. inflammatory cytokines and/or microbial products) tomature into cells capable of activating T cells. An inhibitor ofdeoxyhypusine synthase, the enzyme required to activate eIF5A, was foundto inhibit T lymphocyte activation by dendritic cells by preventing CD83surface expression (Kruse et al., 2000). Thus, eIF5A may facilitatedendritic cell maturation by acting as a nucleocytoplasmic shuttle forCD83 mRNA.

In both of these studies (Bevec et al., 1994; Kruse et al., 2000)implicating a role for eIF5A in the immune system, the authors did notspecify nor identify which isoform of eIF5A they were examining, nor didthey have a reason to. As discussed above, humans are known to have twoisoforms of eIF5A, apoptosis-specific eIF-5A (“eIF5A1”) andproliferating eIF-5A (“eIF-5A2”), both encoded on separate chromosomes.Prior to the present inventors discoveries it was believed that both ofthese isoforms were functional redundant. The oligonucleotide describedby Bevec et al., that was used to detect eIf5A mRNA in stimulated PBMCshad 100% homology to human apoptosis-specific eIF-5A and the studypre-dates the cloning of proliferating &IF-5A. Similarly, the primersdescribed by Kruse et al. that were used to detect eIF5A by reversetranscription polymerase chain reaction during dendritic cell maturationhad 100% homology to human apoptosis-specific eIF-5A.

The present invention relates to controlling the expression ofapoptosis-specific eIF-5A to control the rate of dendritic cellmaturation and PBMC activation, which in turn may control the rate of Tcell-mediated immunity. The present inventors studied the role ofapoptosis-specific eIF-5A in the differentiation of monocytes intoadherent macrophages using the U-937 cell line, as U-937 is known toexpress eIF-5A mRNA (Bevec et al., 1994). U-937 is a human monocyte cellline that grows in suspension and will become adherent and differentiateinto macrophages upon stimulation with PMA. When PMA is removed bychanging the media, the cells become quiescent and are then capable ofproducing cytokines (Barrios-Rodiles et al., J. Immunol., 163:963-969(1999)). In response to lipopolysaccharide (LPS), a factor found on theouter membrane of many bacteria known to induce a general inflammatoryresponse, the macrophages produce both TNF-α and IL-1β (Barrios-Rodileset al., 1999). See FIG. 78 showing a chart of stem cell differentiationand the resultant production of cytokines. The U-937 cells also produceIL-6 and IL-10 following LPS-stimulation (Izeboud et al., J. Receptor &Signal Transduction Research, 19(1-4):191-202. (1999)).

Using U-937 cells, it was shown that apoptosis-specific eIF-5A isupregulated during monocyte differentiation and TNF-α secretion. SeeFIG. 77. Accordingly, one aspect of the invention provides for a methodof inhibiting or delaying maturation of macrophages to inhibit or reducethe production of cytokines. This method involves providing an agentthat is capable of reducing the expression of either DHS orapoptosis-specific eIF-5A. By reducing or eliminating expression of DHS,apoptosis-specific eIF-5A activation will be reduced or eliminated.Since, apoptosis-specific eIF-5A is upregulated during monocytedifferentiation and TNF-α secretion, it is believed that it is necessaryfor these events to occur. Thus, by reducing or eliminating activationof apoptosis-specific eIF-5A or by directly reducing or eliminatingapoptosis-specific eIF-5A expression, monocyte differentiation and TNF-αsecretion can be reduced or eliminated. Any agent capable of reducingthe expression of DHS or apoptosis-specific eIF-5A may be used andincludes, but is not limited to antisense oligonucleotides or siRNAs asdescribed herein.

The present inventors have studied the ability of humanapoptosis-specific eIF-5A to promote translation of cytokines by actingas a nucleocytoplasmic shuttle for cytokine mRNAs in vitro using a cellline known to predictably produce cytokine(s) in response to a specificstimulus. Some recent studies have found that human liver cell lines canrespond to cytokine stimulation by inducing production of othercytokines. HepG2 is a well characterized human hepatocellular carcinomacell line found to be sensitive to cytokines. In response to IL-1β,HepG2 cells rapidly produce TNF-α mRNA and protein in a dose-dependentmanner (Frede et al., 1996; Rowell et al., 1997; Wordemann et al.,1998). Thus, HepG2 cells were used as a model system to study theregulation of TNF-α production. The present inventors have shown thatinhibition of human apoptosis-specific eIF-5A expression in HepG2 cellscaused the cells to produce less TNF-α after having been transfectedwith antisense oligonucleotide of directed toward apoptosis factor 5A.

Thus one embodiment of the present invention provides a method forreducing levels of a cytokine. The method involves administering anagent capable of reducing expression of apoptosis factor 5A1. Reducingexpression of apoptosis factor 5A1 also reduces expression of thecytokine and thus leads to a decreased amount of the cytokine producedby cell. The cytokine is a preferably a pro-inflammatory cytokine,including, but not limited to IL-1, IL-18, IL-6 and TNF-α.

Antisense oligonucleotides are as discussed above. Exemplary antisenseoligonucleotides of human apoptosis-specific eIF-5A are selected fromthe group consisting of SEQ ID NO: 35, 37, and 39 or is an antisensenucleotide that hybridizes under highly stringent conditions to asequence selected from the group consisting of SEQ ID NO: 35, 37, and39.

An agent may also comprise a siRNA of human apoptosis-specific eIF-5Aand are as discussed above. Exemplary siRNAs have a sequence selectedfrom the group consisting of SEQ ID NO: 30, 31, 32 and 33 or is a siRNAthat hybridizes under highly stringent conditions to a sequence selectedfrom the group consisting of SEQ ID NO: 30, 31, 32 and 33. FIGS. 65-67show that cells transfected with human apoptosis-specific eIF-5A siRNAshave a lower percentage of cells undergoing apoptosis after exposure toamptothecin and TNF-α.

The present invention is also directed to a method for reducing theexpression of p53. This method involves administering an agent capableof reducing expression of apoptosis factor 5A, such as the antisenseoligonucleotides or the siRNAs described above. Reducing expression ofapoptosis-specific eIF-5A reduces expression of p53 as shown in FIG. 52and example 10.

The present invention is also directed to a method for increasing theexpression of Bcl-2. This method entails administering an agent capableof reducing expression of human apoptosis-specific eIF-5A. Preferredagents include antisense oligonucleotides and siRNAs described above.Reducing expression of apoptosis-specific eIF-5A increases expression ofBcl-2 as shown in FIG. 63 and example 13. FIG. 63 shows that cellstransfected with apoptosis-specific eIF-5A siRNA produced lessapoptosis-specific eIF-5A protein and in addition, produced more Bcl-2protein. A decrease in apoptosis-specific eIF-5A expression correlateswith an increase in BCL-2 expression.

The present invention also provides a method for reducing levels ofTNF-alpha in a patient in need thereof comprising administering to saidpatient either antisense oligonucleotide or siRNAs of apoptosis-specificeIF-5A as described above. As demonstrated in FIG. 69 and example 14,cells transfected with antisense apoptosis-specific eIF-5Aoligonucleotides of the present invention produced less TNF-α afterinduction with IL-1 than cells not transfected with such antisenseoligonucleotides.

Further, the present invention provides a method of treatingpathological conditions characterized by an increased IL-1, TNF-alpha,IL-61 or IL-18 level comprising administering to a mammal having saidpathological condition, agents to reduce expression ofapoptosis-specific eIF-5A as described above (antisense oligonucleotidesand siRNA).

Known pathological conditions characterized by an increase in IL-1,TNF-alpha, or Il-6 levels include, but are not limited toarthritis-rheumatoid and osteo arthritis, asthma, allergies, arterialinflammation, crohn's disease, inflammatory bowel disease, (ibd),ulcerative colitis, coronary heart disease, cystic fibrosis, diabetes,lupus, multiple sclerosis, graves disease, periodontitis, glaucoma &macular degeneration, ocular surface diseases including keratoconus,organ ischemia-heart, kidney, repurfusion injury, sepsis, multiplemyeloma, organ transplant rejection, psoriasis and eczema. Thus,reducing expression of apoptosis-specific eIF-5A with the antisenseoligonucleotides, siRNAs and methods of the present invention, mayprovide relief from these pathological conditions.

The present invention also provides a method of delivering siRNA tomammalian lung cells in vivo. siRNAs directed against apoptosis-specificeIF-5A were administered intranasally (mixed with water) to mice. 24hours after administration of the siRNA against apoptosis-specificeIF-5A, lipopolysaccharide (LPS) was administered intranasally to themice. After another 24 hours, the right lung was removed andmyeloperoxidase was measured. The mouse apoptosis-specific eIF-5A siRNAsuppressed myeloperoxidase by nearly 90% as compared to the controlsiRNA. In the study, there were 5 mice in each group. The results ofthis study show that siRNA can be delivered successfully in vivo to lungtissue in mammals, and that siRNA directed against apoptosis-specificeIF-5A inhibits the expression of apoptosis-specific eIF-5A resulting ina suppression of myeloperoxidase production. Myeloperoxidase (“MPO”) isa lysosomal enzyme that is found in neutrophils. The myeloperoxidase isan enzyme that uses hydrogen peroxidase to convert chloride tohypochlorous acid. The hypochlorous acid reacts with and destroysbacteria. Myeloperoxidase is also produced when arteries are inflamed.Thus, it is clear that myeloperoxidase is associated with neutrophilsand the inflammation response. The present inventors have shown that bydown regulating apoptosis-specific eIF-5A with siRNAs shows a markeddecrease in the myeloperoxidase in lung tissue after exposed to LPS(which normally produces an inflammatory response involving theproduction of myeloperoxidase). Thus, the present inventors have shownthat using siRNAs against apoptosis-specific eIF-5A can decrease orsuppress the amount of myeloperoxidase in lung tissue and thus decreaseor suppress the inflammation response.

LPS is a macromolecular cell surface antigen of bacteria that whenapplied in vivo triggers a network of inflammatory responses.Intranasally delivering LPS causes an increase in the number ofneutrophils in the lungs. One of the primary events is the activation ofmononuclear phagocytes through a receptor-mediated process, leading tothe release of a number of cytokines, including TNF-α. In turn, theincreased adherence of neutrophils to endothelial cells induced by TNF-αleads to massive infiltration in the pulmonary space.

Thus, not only have the present inventors shown the correlation betweenapoptosis-specific eIF-5A and the immune response, as well as shown thatsiRNAs against apoptosis-specific eIF-5A an suppress the production ofmyeloperoxidase (i.e. part of the inflammation response). The inventorshave also shown that it is possible to deliver siRNAs in vivo to lungtissue by simple intranasal delivery. The siRNAs were mixed only inwater. This presents a major breakthrough and discovery as othersskilled in the art have attempted to design acceptable delivery methodsfor siRNA.

The ability to reduce inflammation is of direct importance in manydiseases. MPO levels are a critical predictor of heart attacks andcytokine-induced inflammation caused by autoimmune disorders. Thisability to decrease or suppress the inflammation response may serveuseful in treating inflammation related disorders such as auto immunedisorders. In addition, the ability to lower MPO could be a means ofprotecting patients from ischemic events and heart attacks.

In another experiment, mice were similarly treated with siRNAs directedagainst apoptosis-specific eIF-5A. Lipopolysaccharide (LPS) wasadministered to the mice to induce inflammation and an immune systemresponse. Under control conditions, LPS kills thymocytes, which areimportant immune system precursor cells created in the thymus to fendoff infection. However, using the siRNAs directed againstapoptosis-specific eIF-5A allowed approximately 90% survivability of thethymocytes in the presence of LPS. When thymocytes are destroyed, sincethey are precursors to T cells, the body's natural immunity iscompromised by not being able to produce T cells and thus can't ward offbacterial infections and such. Thus, using the siRNAs againstapoptosis-specific eIF-5A can be used to reduce inflammation (as shownby a lower level of MPO in the first example) without destroying thebody's natural immune defense system.

One embodiment of the present invention relates to reducing NFk beta(“NFkB”) levels by inhibiting apoptosis-specific eIF-5A with siRNAstargeted at apoptosis-specific eIF-5A. NFk beta is a majorcell-signaling molecule for inflammation—its activation induces theexpression of COX-2, which leads to tissue inflammation. The expressionof the COX-2-encoding gene, believed to be responsible for the massiveproduction of prostaglandins at inflammatory sites, is transcriptionallyregulated by NFkB. NFkB resides in the cytoplasm of the cell and isbound to its inhibitor. Injurious and inflammatory stimuli release NFkBfrom the inhibitor. NFkB moves into the nucleus and activates the genesresponsible for expressing COX-2. Thus, by reducing levels of NFk beta.,inflammation can be reduced.

In one experiment human epithelial cells (HT-29 cells) were treated withsiRNA targeted at apoptosis-specific eIF-5A. Inflammation was theninduced by NFkB by addition of TINT or interferon gamma and LPS for onehour. The results of this experiment show that inhibiting the expressionof apoptosis-specific eIF-5A with siRNAs provided for a reduction in thelevels of NFkB that were activated by the gamma interferon and LPS. SeeFIG. 74.

One embodiment of the present invention provides methods of inhibitionexpression of endogenous apoptosis-specific eIF-5A in a cell. Inhibitingexpression is preferably carried out by the use of antisensepolynucleotides or siRNAs of apoptosis-specific eIF-5A of the presentinvention described previously. When expression of endogenousapoptosis-specific eIF-5A occurs various effects on the cell result. Forexample, a reduction in expression of the various proteins, factors,receptors, cytokines are seen: p53; pro-inflammatory cytokines (See FIG.112, 113, 114); myeloperoxidase; active NFk beta; TLR4 (See FIG. 109);TNFR-1 (See FIG. 111) and iNOS. A reduction in expression of endogenousapoptosis-specific eIF-5A increases levels expression of Bcl-2 in thecell.

It is understood that the antisense nucleic acids siRNAs of the presentinvention, where used in an animal for the purpose of prophylaxis ortreatment, will be administered in the form of a compositionadditionally comprising a pharmaceutically acceptable carrier. Suitablepharmaceutically acceptable carriers include, for example, one or moreof water, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. Pharmaceuticallyacceptable carriers can further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the bindingproteins. The compositions of the injection can, as is well known in theart, be formulated so as to provide quick, sustained or delayed releaseof the active ingredient after administration to the mammal.

The compositions of this invention can be in a variety of forms. Theseinclude, for example, solid, semi-solid and liquid dosage forms, such astablets, pills, powders, liquid solutions, dispersions or suspensions,liposomes, suppositories, injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application.

Such compositions can be prepared in a manner well known in thepharmaceutical art. In making the composition the active ingredient willusually be mixed with a carrier, or diluted by a carrier, and/orenclosed within a carrier which can, for example, be in the form of acapsule, sachet, paper or other container. When the carrier serves as adiluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, excipient or medium for the active ingredient. Thus, thecomposition can be in the form of tablets, lozenges, sachets, cachets,elixirs, suspensions, aerosols (as a solid or in a liquid medium),ointments containing for example up to 10% by weight of the activecompound, soft and hard gelatin capsules, suppositories, injectionsolutions, suspensions, sterile packaged powders and as a topical patch.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration. The Examples are set forth to aidin understanding the invention but are not intended to, and should notbe construed to, limit its scope in any way. The examples do not includedetailed descriptions of conventional methods. Such methods are wellknown to those of ordinary skill in the art and are described innumerous publications. Detailed descriptions of conventional methods,such as those employed in the construction of vectors and plasmids, theinsertion of nucleic acids encoding polypeptides into such vectors andplasmids, the introduction of plasmids into host cells, and theexpression and determination thereof of genes and gene products can beobtained from numerous publication, including Sambrook, J. et al.,(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Laboratory Press. All references mentioned herein areincorporated in their entirety.

EXAMPLES Example 1 Visualization of Apoptosis in Rat Corpus Luteum byDNA Laddering

The degree of apoptosis was determined by DNA laddering. Genomic DNA wasisolated from dispersed corpus lineal cells or from excised corpusluteum tissue using the QIAamp DNA Blood Kit (Qiagen) according to themanufacturer's instructions. Corpus luteum tissue was excised before theinduction of apoptosis by treatment with PGF-2α, 1 hour and 24 hoursafter induction of apoptosis. The isolated DNA was end-labeled byincubating 500 ng of DNA with 0.2 μCi [α-³²P]dCTP, 1 mM Tris, 0.5 mMEDTA, 3 units of Klenow enzyme, and 0.2 pM each of dATP, dGTP, and dTTPat room temperature for 30 minutes. Unincorporated nucleotides wereremoved by passing the sample through a 1 ml Sepadex G-50 columnaccording to Sambrook et al. The samples were then resolved byTris-acetate-EDTA (1.8%) gel electrophoresis. The gel was dried for 30minutes at room temperature under vacuum and exposed to x-ray film at−80° C. for 24 hours.

In one experiment, the degree of apoptosis in superovulated rat corpuslutea was examined either 0, 1, or 24 hours after injection with PGF-2α.In the 0 hour control, the ovaries were removed without PGF-2αinjection. Laddering of low molecular weight DNA fragments reflectingnuclease activity associated with apoptosis is not evident in controlcorpus luteum tissue excised before treatment with PGF-2α, but isdiscernible within 1 hour after induction of apoptosis and is pronouncedby 24 hours after induction of apoptosis, which is shown in FIG. 16. Inthis figure, the top panel is an autoradiograph of the Northern blotprobed with the ³²P-dCTP-labeled 3′-untranslated region of rat corpusluteum apoptosis-specific DHS cDNA. The lower panel is the ethidiumbromide stained gel of total RNA. Each lane contains 10 μg RNA. The dataindicate that there is down-regulation of apoptosis-specific transcriptfollowing serum withdrawal.

In another experiment, the corresponding control animals were treatedwith saline instead of PGF-2α. Fifteen minutes after treatment withsaline or PGF-2α, corpora lutea were removed from the animals. GenomicDNA was isolated from the corpora lutea at 3 hours and 6 hours afterremoval of the tissue from the animals. DNA laddering and increased endlabeling of genomic DNA are evident 6 hours after removal of the tissuefrom the PGF-2α-treated animals, but not at 3 hours after removal of thetissue. See FIG. 17. DNA laddering reflecting apoptosis is also evidentwhen corpora lutea are excised 15 minutes after treatment with PGF-2αand maintained for 6 hours under in vitro conditions in EBSS (Gibco).Nuclease activity associated with apoptosis is also evident from moreextensive end labeling of genomic DNA.

In another experiment, superovulation was induced by subcutaneousinjection with 500 μg of PGF-2α. Control rats were treated with anequivalent volume of saline solution. Fifteen to thirty minutes later,the ovaries were removed and minced with collagenase. The dispersedcells from rats treated with PGF-2α and were incubated in 10 mmglutamine+10 mm spermidine for 1 hour and for a further 5 hours in 10min glutamine without spermidine (lane 2) or in 10 mm glutamine+10 mmspermidine for 1 hour and for a further 5 hours in 10 mm glutamine+1 mmspermidine (lane 3). Control cells from rats treated with saline weredispersed with collagenase and incubated for 1 hour and a thither 5hours in glutamine only (lane 1). Five hundred nanograms of DNA fromeach sample was labeled with [α-³²P]-dCTP using klenow enzyme, separatedon a 1.8% agarose gel, and exposed to film for 24 hours. Results areshown in FIG. 18.

In yet another experiment, superovulated rats were injectedsubcutaneously with 1 mg/100 g body weight of spermidine, delivered inthree equal doses of 0.333 mg/100 g body weight, 24, 12, and 2 hoursprior to a subcutaneous injection with 500 μg PGF-2α. Control rats weredivided into three sets: no injections, three injections of spermidinebut no PGF-2α; and three injections with an equivalent volume of salineprior to PGF-2α, treatment. Ovaries were removed front the rats either 1hour and 35 minutes or 3 hours and 45 minutes after prostaglandintreatment and used for the isolation of DNA. Five hundred nanograms ofDNA from each sample was labeled with [α-³²P]-dCTP using Klenow enzyme,separated on a 1.8% agarose gel, and exposed to film for 24 hours: lane1, no injections (animals were sacrificed at the same time as for lanes3-5); lane 2, three injections with spermidine (animals were sacrificedat the same time as thr lanes 3-5); lane 3, three injections with salinefollowed by injection with PGF-2α (animals were sacrificed 1 h and 35min after treatment with PGF-2α); lane 4, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 1h and 35 min after treatment with PGF-2α); lane 5, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 1h and 35 min after treatment with PGF-2α); lane 6, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 3h and 45 min after treatment with PGF-2α); lane 7, three injections withspermidine followed by injection with PGF-2α (animals were sacrificed 3h and 45 min after treatment with PGF-2α). Results are shown in FIG. 19.

RNA Isolation

Total RNA was isolated from corpus luteum tissue removed from rats atvarious times after PGF-2α induction of apoptosis. Briefly, the tissue(5 g) was ground in liquid nitrogen. The ground powder was mixed with 30ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mM NaOAc pH8.5, 0.8% β-mercaptoethanol). The mixture was filtered through fourlayers of Miracloth and centrifuged at 10,000 g at 4° C. for 30 minutes.The supernatant was then subjected to cesium chloride density gradientcentrifugation at 11,200 g for 20 hours. The pelleted RNA was rinsedwith 75% ethanol, resuspended in 600 ml DEPC-treated water and the RNAprecipitated at −70° C. with 1.5 ml 95% ethanol and 60 ml of 3M NaOAc.

Genomic DNA Isolation and Laddering

Genomic DNA was isolated from extracted corpus luteum tissue ordispersed corpus luteal cells using the QIAamp DNA Blood Kit (Qiagen)according to the manufacturer's instructions. The DNA was end-labeled byincubating 500 ng of DNA with 0.2 μCi [α-³²P]dCTP, 1 mM Tris, 0.5 mMEDTA, 3 units of Klenow enzyme, and 0.2 pM each of dATP, dGTP, and dTTP,at room temperature for 30 minutes. Unincorporated nucleotides wereremoved by passing the sample through a 1-ml Sephadex G-50 columnaccording to the method described by Maniatis et al. The samples werethen resolved by Tris-acetate-EDTA (2%) gel electrophoresis. The gel wasdried for 30 minutes at room temperature under vacuum and exposed tox-ray film at −80° C. for 24 hours.

Plasmid DNA Isolation, DNA Sequencing

The alkaline lysis method described by Sambrook et al., supra, was usedto isolate plasmid DNA. The full-length positive cDNA clone wassequenced using the dideoxy sequencing method. Sanger et al., Proc.Natl. Acad. Sci. LISA, 74:5463-5467. The open reading frame was compiledand analyzed using BLAST search (GenBank, Bethesda, Md.) and sequencealignment was achieved using a BCM Search Launcher: Multiple SequenceAlignments Pattern-Induced Multiple Alignment Method (see F. Corpet,Nuc. Acids Res., 16:10881-10890, (1987). Sequences and sequencealignments are shown in FIGS. 5-11.

Northern Blot Hybridization of Rat Corpus Luteum RNA

Twenty milligrams of total RNA isolated from rat corpus luteum atvarious stages of apoptosis were separated on 1% denatured formaldehydeagarose gels and immobilized on nylon membranes. The full-length ratapoptosis-specific eIF-5A cDNA (SEQ ID NO:1) labeled with ³²P-dCTP usinga random primer kit (Boehringer) was used to probe the membranes 7×10⁷.Alternatively, full length rat DHS cDNA (SEQ NO:6) labeled with ³²P-dCTPusing a random primer kit (Boehringer) was used to probe the membranes(7×10⁷ cpm). The membranes were washed once with 1×SSC, 0.1% SDS at roomtemperature and three times with 0.2×SSC, 0.1% SDS at 65° C. Themembranes were dried and exposed to X-ray film overnight at −70° C.

As can be seen, apoptosis-specific eIF-5A and DHS are both upregulatedin apoptosing corpus luteum tissue. Expression of apoptosis-specificeIF-5A is significantly enhanced after induction of apoptosis bytreatment with PGF-2α—low at time zero, increased substantially within 1hour of treatment, increased still more within 8 hours of treatment andincreased slightly within 24 hours of treatment (FIG. 14). Expression ofDHS was low at time zero, increased substantially within 1 hour oftreatment, increased still more within 8 hours of treatment andincreased again slightly within 24 hours of treatment (FIG. 15).

Generation of an Apoptosing Rat Corpus Luteum RT-PCR Product UsingPrimers Based on Yeast, Fungal and Human eIF-54 Sequences

A partial-length apoptosis-specific eIF-5A sequence (SEQ ID NO:11)corresponding to the 3′ end of the gene was generated from apoptosingrat corpus luteum RNA template by RT-PCR using a pair of oligonucleotideprimers designed from yeast, fungal and human apoptosis-specific eIF-5Asequences. The upstream primer used to isolate the 3′ end of the ratapoptosis-specific eIF-5A gene is a 20 nucleotide degenerate primer: 5′TCSAARACHGGNAAGCAYGG 3′ (SEQ ID NO:9), wherein S is selected from C andG; R is selected from A and G; H is selected from A, T, and C; Y isselected from C and T; and N is any nucleic acid. The downstream primerused to isolate the 3′ end of the rat eIF-5A gene contains 42nucleotides: 5′ GCGAAGCTTCCATGG CTCGAGTTTTTTTTTTTTTTTTTTTTT 3° (SEQ EDNO:10). A reverse transcriptase polymerase chain reaction (RT-PCR) wascarried put. Briefly, using 5 mg of the downstream primer, a firststrand of cDNA was synthesized. The first strand was then used as atemplate in a RT-PCR using both the upstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence a 900 by fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LaJolla, Calif.) using blunt end ligationand sequenced (SEQ ID NO:11). The cDNA sequence of the 3′ end is SEQ IDNO:11 and the amino acid sequence of the 3′ end is SEQ, ID NO:12. SeeFIGS. 1-2.

A partial-length apoptosis-specific eIF-5A sequence (SEQ ID NO: 15)corresponding to the 5 end of the gene and overlapping with the 3′ endwas generated from apoptosing rat corpus luteum RNA template by RT-PCR.The 5° primer is a 24-mer having the sequence, 5′CAGGTCTAGAGTTGGAATCGAAGC 3′ (SEQ ID NO:13), that was designed from humaneIF-5A sequences. The 3° primer is a 30-mer having the sequence, 5°ATATCTCGAGCCTT GATTGCAACAGCTGCC 3′ (SEQ ID NO:14) that was designedaccording to the 3′ end RT-PCR fragment. A reversetranscriptase-polymerase chain reaction (RT-PCR) was carried out.Briefly, using 5 mg of the downstream primer, a first strand of cDNA wassynthesized. The first strand was then used as a template in a RT-PCRusing both the upstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence a 500 by fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LaJolla, Calif.) using XhaI and XhoIcloning sites present in the upstream and downstream primers,respectively, and sequenced (SEQ ID NO:15). The cDNA sequence of the 5′end is SEQ ID NO:15, and the amino acid sequence of the 5′ end is SEQ IDNO:16. See FIG. 2.

The sequences of the 3′ and 5′ ends of the rat apoptosis-specific eIF-5A(SEQ ID NO:11 and SEQ ID NO:15, respectively) overlapped and gave riseto the full-length cDNA sequence (SEQ ID NO:1). This full-lengthsequence was aligned and compared with sequences in the GeneBank database. See FIGS. 1-2. The cDNA clone encodes a 154 amino acid polypeptide(SEQ IT) NO:2) having a calculated molecular mass of 16.8 KDa. Thenucleotide sequence, SEQ ID NO:1, for the full length cDNA of the ratapoptosis-specific corpus luteum eIF-5A gene obtained by RT-PCR isdepicted in FIG. 3 and the corresponding derived amino acid sequence isSEQ ID NO:9. The derived full-length amino acid sequence of eIF-5A wasaligned with human and mouse eIF-5a sequences. See FIG. 7-9.

Generation of an Apoptosing Rat Corpus Luteum RT-PCR Product UsingPrimers Based on a Human DHS Sequence

A partial-length DHS sequence (SEQ ID NO:6) corresponding to the 3′ endof the gene was generated from apoptosing rat corpus luteum RNA templateby RT-PCR using a pair of oligonucleotide primers designed from a humanDHS sequence. The 5′ primer is a 20-mer having the sequence, 5′GTCTGTGTArITATTGGGCCC 3′ (SEQ ID NO. 17); the 3 primer is a 42-merhaving the sequence, 5′ GCGAAGCTTCCATGGC TCGAGTTTTTTTTTTTTITTTTT 3′ (SEQID NO:18). A reverse transcriptase polymerase chain reaction (RT-PCR)was carried out. Briefly, using 5 mg of the downstream primer, a firststrand of cDNA was synthesized. The first strand was then used as atemplate in a RT-PCR using both the upstream and downstream primers.

Separation of the RT-PCR products on an agarose gel revealed thepresence a 606 by fragment, which was subcloned into pBluescript™(Stratagene Cloning Systems, LaJolla, Calif.) using blunt end ligationand sequenced (SEQ ID NO:6). The nucleotide sequence (SEQ ID NO:6) forthe partial length cDNA of the rat apoptosis-specific corpus luteum DHSgene obtained by RT-PCR is depicted in FIG. 4 and the correspondingderived amino acid sequence is SEQ ID NO.7.

Isolation of Genomic DNA and Southern Analysis

Genomic DNA for southern blotting was isolated from excised rat ovaries.Approximately 100 mg of ovary tissue was divided into small pieces andplaced into a 15 ml tube. The tissue was washed twice with 1 ml of PBSby gently shaking the tissue suspension and then removing the PBS usinga pipette. The tissue was resuspended in 2.06 ml of DNA-buffer (0.2 MTris-HCl pH 8.0 and 0.1 mM EDTA) and 240 μl of 10% SDS and 100 μl ofproteinase K (Boehringer Martheim; 10 mg/ml) was added. The tissue wasplaced in a shaking water bath at 45° C. overnight. The following dayanother 100 μl of proteinase K (10 mg/ml) was added and the tissuesuspension was incubated in a water-bath at 45° C. for an additional 4hours. After the incubation the tissue suspension was extracted oncewith an equal volume of phenol:chloroform:iso-amyl alcohol (25:24:1) andonce with an equal volume of chloroform:iso-amyl alcohol (24:1).Following the extractions 1/10th volume of 3M sodium acetate (pH 5.2)and 2 volumes of ethanol were added. A glass pipette sealed and formedinto a hook using a Bunsen burner was used to pull the DNA threads outof solution and to transfer the DNA into a clean microcentrifuge tube.The DNA was washed once in 70% ethanol and air-dried for 10 minutes. TheDNA pellet was dissolved in 500 μl of 10 mM Tris-HCl (pH 8.0), 10 μl ofRNase A (10 mg/ml) was added, and the DNA was incubated for 1 hour at37° C. The DNA was extracted once with phenol:chloroform:iso-amylalcohol (25:24:1) and the DNA was precipitated by adding 1/10th volumeof 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. The DNA waspelleted by centrifugation for 10 minutes at 13,000×g at 4° C. The DNApellet was washed once in 70% ethanol and dissolved in 200 μl 10 mMTris-HCl (pH 8.0) by rotating the DNA at 4° C. overnight.

For Southern blot analysis, genomic DNA isolated from rat ovaries wasdigested with various restriction enzymes that either do not cut in theendogenous gene or cut only once. To achieve this, 10 μg genomic DNA, 20μl 10× reaction buffer and 100 U restriction enzyme were reacted forfive to six hours in a total reaction volume of 200 μl. Digested DNA wasloaded onto a 0.7% agarose gel and subjected to electrophoresis for 6hours at 40 volts or overnight at 15 volts. After electrophoresis, thegel was depurinated for 10 minutes in 0.2 N HCl followed by two15-minute washes in denaturing solution (0.5 M NaOH, 1.5 M NaCl) and two15 minute washes in neutralizing buffer (1.5 M NaCl, 0.5 M Tris-HCl pH7.4). The DNA was transferred to a nylon membrane, and the membrane wasprehybridized in hybridization solution (40% formamide, 6×SSC,5×Denhardt's, solution (1×Denhardt's solution is 0.02% Ficoli, 0.02%PVP, and 0.02% BSA), 0.5% SDS, and 1.5 mg of denatured salmon spermDNA). A 700 by PCR fragment of the 3 UTR of rat eIF-5A cDNA (650 by of3° UTR and 50 by of coding) was labeled with [a-32P]-dCTP by randompriming and added to the membrane at 1×106

Similarly, a 606 by PCR fragment of the rat DHS cDNA (450 by coding and156 by 3′ UTR) was random prime labeled with [α-³²P]-dCTP and added at1×10 6 cpm/ml to a second identical membrane. The blots were hybridizedovernight at 42° C. and then washed twice with 2×SSC and 0.1% SDS at 42°C. and twice with 1×SSC and 0.1% SDS at 42° C. The blots were thenexposed to film for 3-10 days.

Rat corpus genomic DNA was cut with restriction enzymes as indicated onFIG. 20 and probed with ³²P-dCTP-labeled full-length eIF-5A cDNA.Hybridization under high stringency conditions revealed hybridization ofthe full-length cDNA probe to several restriction fragments for eachrestriction enzyme digested DNA sample, indicating the presence ofseveral isoforms of eIF-5A. Of particular note, when rat genomic DNA wasdigested with EcoRV, which has a restriction site within the openreading frame of apoptosis-specific eIF-5A, two restriction fragments ofthe apoptosis-specific isoform of eIF-5A were detectable in the Southernblot. The two fragments are indicated with double arrows in FIG. 20. Therestriction fragment corresponding to the apoptosis-specific isoform ofeIF-5A is indicated by a single arrow in the lanes labeled EcoR1 andBamH1, restriction enzymes for which there are no cut sites within theopen reading frame. These results suggest that the apoptosis-specificeIF-5A is a single copy gene in rat. As shown in FIGS. 5 through 13, theeIF-5A gene is highly conserved across species, and so it would beexpected that there is a significant amount of conservation betweenisoforms within any species.

FIG. 21 shows a Southern blot of rat genomic DNA probed with³²P-dCFP-labeled partial-length rat corpus luteum DHS cDNA. The genomicDNA was cut with EcoRV, a restriction enzyme that does not cut thepartial-length cDNA used as a probe. Two restriction fragments areevident indicating that there are two copies of the gene or that thegene contains an intron with an EcoRV site.

Example 2

The present example demonstrates modulation of apoptosisapoptosis-specific eIF-5A (increasing apoptosis with apoptosis-specificeIF-5A in sense orientation)

Culturing of COS-7 Cells and Isolation of RNA

COS-7, an African green monkey kidney fibroblast-like cell linetransformed with a mutant of SV40 that codes for wild-type T antigen,was used for all transfection-based experiments. COS-7 cells werecultured in Dulbecco's Modified Eagle's medium (MEM) with 0.584 gramsper liter of L-glutamine, 4.5 g of glucose per liter, and 0.37% sodiumbicarbonate. The culture media was supplemented with 10% fetal bovineserum (FBS) and 100 units of penicillin/streptomycin. The cells weregrown at 37° C. in a humidified environment of 5% CO₂ and 95% air. Thecells were subcultured every 3 to 4 days by detaching the adherent cellswith a solution of 0.25% trypsin and 1 mM EDTA. The detached cells weredispensed at a split ratio of 1:10 in a new culture dish with freshmedia.

COS-7 cells to be used for isolation of RNA were grown in 150-mm tissueculture treated dishes (Corning). The cells were harvested by detachingthem with a solution of trypsin-EDTA. The detached cells were collectedin a centrifuge tube, and the cells were pelleted by centrifugation at3000 rpm for 5 minutes. The supernatant was removed, and the cell pelletwas flash-frozen in liquid nitrogen. RNA was isolated from the frozencells using the GenElute Mammalian Total RNA Miniprep kit (Sigma)according to the manufacturer's instructions.

Construction of Recombinant Plasmids and Transfection of COS-7 Cells

Recombinant plasmids carrying the full-length coding sequence of ratapoptosis-specific eIF-5A in the sense orientation and the 3untranslated region (UTR) of rat apoptosis-specific eIF-5A in theantisense orientation were constructed using the mammalian epitope tagexpression vector, pHM6 (Roche Molecular Biochemicals), which isillustrated in FIG. 21. The vector contains the following: CMVpromoter—human cytomegalovirus immediate-early promoter/enhancer;HA—nonapeptide epitope tag from influenza hemagglutinin; BGH pA—Bovinegrowth hormone polyadenylation signal; f1 ori—f1 origin; SV40 on SV40early promoter and origin; Neomycin—Neomycin resistance (0418) gene;SV40 pA SV40 polyadenylation signal; Col E1—ColE1 origin;Ampicillin—Ampicillin resistance gene. The full-length coding sequenceof rat apoptosis-specific eIF-5A and the 3° UTR of ratapoptosis-specific eIF-5A were amplified by PCR from the original rateIF-5A RT-PCR fragment in pBluescript (SEQ ID NO:1). To amplify thefull-length eIF-5A the primers used were as follows: Forward 5′GCCAAGCTTAATGGCAGATGATTT GG 3′ (SEQ ID NO: 59) (Hind3) and Reverse 5°CTGAATTCCAGT TATTITGCCNI′GG 3′ (SEQ U) NO:60) (EcoR1). To amplify the 3′UTR rat apoptosis-specific eIF-5A the primers used were as follows:forward 5° AATGAATTCCGCCATGACAGAGGAGGC 3′ (SEQ ID NO: 61) (EcoR1) andreverse 5═ GCGAAGCTTCCATGGCTCGAGTTTTTTFITITITTITTTTIT 3′ (SEQ ID NO: 62)(Hind3).

The full-length rat apoptosis-specific eIF-5A PCR product isolated afteragarose gel electrophoresis was 430 by in length while the 3′ UTR ratapoptosis-specific PCR product was 697 by in length. Both PCR productswere subcloned into the Hind 3 and EcoR1 sites of pHM6 to createpHM6-full-length apoptosis-specific eIF-5A and pHM6-antisense3′UTReIF-5A. The full-length rat apoptosis-specific eIF-5A PCR productwas subcloned in frame with the nonapeptide epitope tag from influenzahemagglutinin (HA) present upstream of the multiple cloning site toallow for detection of the recombinant protein using ananti-[HA]-peroxidase antibody. Expression is driven by the humancytomegalovirus immediate-early promoter/enhancer to ensure high levelexpression in mammalian cell lines. The plasmid also features aneomycin-resistance (G418) gene, which allows for selection of stabletransfectants, and a SV40 early promoter and origin, which allowsepisomal replication in cells expressing SV40 large antigen, such asCOS-7.

COS-7 cells to be used in transfection experiments were cultured ineither 24 well cell culture plates (Corning) for cells to be used forprotein extraction, or 4 chamber culture slides (Falcon) for cells to beused for staining. The cells were grown in DMEM media supplemented with10% FBS, but lacking penicillin/streptomycin, to 50 to 70% confluency.Transfection medium sufficient for one well of a 24-well plate orculture slide was prepared by diluting 0.32 μg of plasmid DNA in 42.5 μlof serum-free DMEM and incubating the mixture at room temperature for 15minutes. 1.6 μl of the transfection reagent, LipofectAMINE (Gibco, BRL),was diluted in 42.5 μl of serum-free DMEM and incubated fix 5 minutes atroom temperature. After 5 minutes the LipofectAMINE mixture was added tothe DNA mixture and incubated together at room temperature for 30 to 60minutes. The cells to be transfected were washed once with serum-freeDMEM before overlaying the transfection medium and the cells were placedback in the growth chamber for 4 hours.

After the incubation, 017 ml of DMEM+20% FBS was added to the cells. Thecells were the cultured for a further 40 hours before either beinginduced to undergo apoptosis prior to staining or harvested for Westernblot analysis. As a control, mock transfections were also performed inwhich the plasmid DNA was omitted from the transfection medium.

Protein Extraction and Western Blotting

Protein was isolated for Western blotting from transfected cells bywashing the cells twice in PBS (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/LNa₂HPO₄, and 0.24 g/L KH₂PO₄) and then adding 150 μl of hot SDSgel-loading buffer (50 mM Tris-HCl pH 6.8, 100 mM dithiothreitol, 2%SDS, 0.1% bromophenol blue, and 10% glycerol). The cell lysate wascollected in a microcentrifuge tube, heated at 95° C. for 10 minutes,and then centrifuged at 13,000×g for 10 minutes. The supernatant wastransferred to a fresh microcentrifuge tube and stored at −20° C. untilready for use.

For Western blotting, 2.5 or 5 μg of total protein was separated on a12% SDS-polyacrylamide gel. The separated proteins were transferred to apolyvinylidene difluoride membrane. The membrane was then incubated forone hour in blocking solution (5% skim milk powder, 0.02% sodium azidein PBS) and washed three times for 15 minutes in PBS-T (PBS+0.05%Tween-20). The membrane was stored overnight in PBS-T at 4° C. Afterbeing warmed to room temperature the next day, the membrane was blockedfor 30 seconds in 1 μg/ml polyvinyl alcohol. The membrane was rinsed 5times in deionized water and then blocked for 30 minutes in a solutionof 5% milk in PBS. The primary antibody was preincubated for 30 minutesin a solution of 5% milk in PBS prior to incubation with the membrane.

Several primary antibodies were used. An anti-[HA]-peroxidase antibody(Roche Molecular Biochemicals) was used at a dilution of 1:5000 todetect expression of the recombinant proteins. Since this antibody isconjugated to peroxidase, no secondary antibody was necessary, and theblot was washed and developed by chemiluminescence. The other primaryantibodies that were used are monoclonal antibodies from Oncogene thatrecognize p53 (Ab-6), Bcl-2 (Ab-1), and c-Myc (Ab-2). The monoclonalantibody to p53 was used at a dilution of 0.1 μg/ml, and the monoclonalantibodies to Bcl-2 and c-Myc were both used at a dilution of 0.83μg/ml. After incubation with primary antibody for 60 to 90 minutes, themembrane was washed 3 times for 15 minutes in PBS-T. Secondary antibodywas then diluted in 1% milk in PBS and incubated with the membrane for60 to 90 minutes. When p53 (Ab-6) was used as the primary antibody, thesecondary antibody used was a goat anti-mouse IgG conjugated to alkalinephosphatase (Rockland) at a dilution of 1:1000. When Bcl-2 (Ab-1) andc-Myc (Ab-2) were used as the primary antibody, a rabbit anti-mouse IgGconjugated to peroxidase (Signia) was used at a dilution of 1:5000.After incubation with the secondary antibody, the membrane was washed 3times in PBS-T.

Two detection methods were used to develop the blots, a colorimetricmethod and a chemiluminescent method. The colorimetric method was usedonly when p53 (Ab-6) was used as the primary antibody in conjunctionwith the alkaline phosphatase conjugated secondary antibody. Boundantibody was visualized by incubating the blot in the dark in a solutionof 0.33 mg/mL nitro blue tetrazolium, 0.165 mg/mL5-bromo-4-chloro-3-indolyl phosphate, 100 mM NaCl, 5 mM MgCl₂, and 100mM Tris-HCl (pH 9.5). The color reaction was stopped by incubating theblot in 2 mM EDTA in PBS. A chemiluminescent detection method was usedfor all other primary antibodies, including anti-[HA]-peroxidase, Bcl-2(Ab-1), and c-Myc (Ab-2). The ECL Plus Western blotting detection kit(Amersham Pharmacia Biotech) was used to detect peroxidase-conjugatedbound antibodies. In brief, the membrane was lightly blotted dry andthen incubated in the dark with a 40:1 mix of reagent A and reagent Bfor 5 minutes. The membrane was blotted dry, placed between sheets ofacetate, and exposed to X-ray film for time periods varying from 10seconds to 10 minutes.

Induction of Apoptosis in COS 7 Cells

Two methods were used to induce apoptosis in transfected COS-7 cells,serum deprivation and treatment with Actinomycin D, streptomyces sp(Calbiochem). For both treatments, the medium was removed 40 hourspost-transfection. For serum starvation experiments, the media wasreplaced with serum- and antibiotic-free DMEM. Cells grown inantibiotic-free DMEM supplemented with 10% FBS were used as a control.For Actinomycin D induction of apoptosis, the media was replaced withantibiotic-free DMEM supplemented with 10% FBS and 1 μg/ml Actinomycin Ddissolved in methanol. Control cells were grown in antibiotic-free DMEMsupplemented with 10% FBS and an equivalent volume of methanol. For bothmethods, the percentage of apoptotic cells was determined 48 hours laterby staining with either Hoescht or Annexin V-Cy3. Induction of apoptosiswas also confirmed by Northern blot analyses, as shown in FIG. 22

Hoescht Staining

The nuclear stain, Hoescht, was used to label the nuclei of transfectedCOS-7 cells in order to identify apoptotic cells based on morphologicalfeatures such as nuclear fragmentation and condensation. A fixative,consisting of a 3:1 mixture of absolute methanol and glacial aceticacid, was prepared immediately before use. An equal volume of fixativewas added to the media of COS-7 cells growing on a culture slide andincubated for 2 minutes. The media/fixative mixture was removed from thecells and discarded, and 1 ml of fixative was added to the cells. After5 minutes the fixative was discarded, and 1 ml of fresh fixative wasadded to the cells and incubated for 5 minutes. The fixative wasdiscarded, and the cells were air-dried for 4 minutes before adding 1 mlof Hoescht stain (0.5 μg/mlHoescht 33258 in PBS). After a 10-minuteincubation in the dark, the staining solution was discarded and theslide was washed 3 times for 1 minute with deionized water. Afterwashing, 1 ml of Mcilvaine's buffer (0.021 M citric acid, 0.058 MNa₂HPO₄.7H₂O; pH 5.6) was added to the cells, and they were incubated inthe dark for 20 minutes. The buffer was discarded, the cells wereair-dried for 5 minutes in the dark and the chambers separating thewells of the culture slide were removed. A few drops of Vectashieldmounting media for fluorescence (Vector Laboratories) was added to theslide and overlaid with a coverslip. The stained cells were viewed undera fluorescence microscope using a UV filter. Cells with brightly stainedor fragmented nuclei were scored as apoptotic.

Annexin F-Cy3 Staining

An Annexin V-Cy3 apoptosis detection kit (Sigma) was used tofluorescently label externalized phosphatidylserine on apoptotic cells.The kit was used according to the manufacturer's protocol with thefollowing modifications, in brief, transfected COS-7 cells growing onfour chamber culture slides were washed twice with PBS and three timeswith 1× Binding Buffer, 150 μl of staining solution (1 μg/ml AnnCy3 in1× Binding Buffer) was added, and the cells were incubated in the darkfor 10 minutes. The staining solution was then removed, and the cellswere washed 5 times with 1× Binding Buffer. The chamber walls wereremoved from the culture slide, and several drops of 1× Binding Bufferwere placed on the cells and overlaid with a coverslip. The stainedcells were analyzed by fluorescence microscopy using a green filter tovisualize the red fluorescence of positively stained (apoptotic) cells.The total cell population was determined by counting the cell numberunder visible light.

Example 3

The present example demonstrates modulation of apoptosisapoptosis-specific eIF-5A.

Using the general procedures and methods described in the previousexamples, FIG. 23 is a flow chart illustrating the procedure fortransient transfection of COS-7 cells, in which cells in serum-freemedium were incubated in plasmid DNA in lipofectAMINE for 4 hours, serumwas added, and the cells were incubated for a further 40 hours. Thecells were then either incubated in regular medium containing serum fora further 48 hours before analysis (i.e. no further treatment), deprivedof serum for 48 hours to induce apoptosis before analysis, or treatedwith actinomycin D for 48 hours to induce apoptosis before analysis.

FIG. 22 is a Western blot illustrating transient expression of foreignproteins in COS-7 cells following transfection with pHM6. Protein wasisolated from COS-7 cells 48 hours after either mock transfection, ortransfection with pHM6-LacZ, pHM6-Antisense 3′ rF5A (pHM6-Antisense 3′UTR rat apoptosis-specific eIF-5A), or pHM6-Sense rF5A (pHM6-Full lengthrat apoptosis-specific eIF-5A. Five μg of protein from each sample wasfractionated by SDS-PAGE, transferred to a PVDF membrane, and Westernblotted with anti-[HA]-peroxidase. The bound antibody was detected bychemiluminescence and exposed to x-ray film for 30 seconds. Expressionof LacZ (lane 2) and of sense rat apoptosis-specific eIF-5A (lane 4) isclearly visible.

As described above, COS-7 cells were either mock transfected ortransfected with pHM6-Sense rF5A (pHM6-Full length ratapoptosis-specific eIF-5A). Forty hours after transfection, the cellswere induced to undergo apoptosis by withdrawal of serum for 48 hours.The caspase proteolytic activity in the transfected cell extract wasmeasured using a fluorometric homogenous easpase assay kit (RocheDiagnostics). DNA fragmentation was also measured using the FragEL DNAFragmentation Apoptosis Detection kit (Oncogene) which labels theexposed 3′-OH ends of DNA fragments with fluorescein-labeleddeoxynucleotides.

Additional COS-7 cells were either mock transfected or transfected withpHM6-Sense rF5A (pHM6-Full length rat apoptosis-specific eIF-5A). Fortyhours after transfection, the cells were either grown for an additional48 hours in regular medium containing serum (no further treatment),induced to undergo apoptosis by withdrawal of serum for 48 hours orinduced to undergo apoptosis by treatment with 0.5 μg/ml of ActinomycinD for 48 hours. The cells were either stained with Hoescht 33258, whichdepicts nuclear fragmentation accompanying apoptosis, or stained withAnnexin V-Cy3, which depicts phosphatidylserine exposure accompanyingapoptosis. Stained cells were also viewed by fluorescence microscopyusing a green filter and counted to determine the percentage of cellsUndergoing apoptosis. The total cell population was counted undervisible light.

FIG. 25 illustrates enhanced apoptosis as reflected by increased caspaseactivity when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation. Expression of rat apoptosis-specific eIF-5A resulted in a60% increase in caspase activity.

FIG. 26 illustrates enhanced apoptosis as reflected by increased DNAfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation. Expression of rat apoptosis-specific eIF-5A resulted in a273% increase in DNA fragmentation. FIG. 27 illustrates detection ofapoptosis as reflected by increased nuclear fragmentation when COS-7cells were transiently transfected with pHM6 containing full-length ratapoptosis-specific eIF-5A in the sense orientation. There is a greaterincidence of fragmented nuclei in cells expressing ratapoptosis-specific eIF-5A. FIG. 28 illustrates enhanced apoptosis asreflected by increased nuclear fragmentation when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-specific eIF-5A in the sense orientation. Expression of ratapoptosis-specific eIF-5A resulted in a 27% and 63% increase in nuclearfragmentation over control in non-serum starved and serum starvedsamples, respectively.

FIG. 29 illustrates detection of apoptosis as reflected byphosphatidylserine exposure when COS-7 cells were transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation. FIG. 30 illustrates enhanced apoptosisas reflected by increased phosphatidylserine exposure when COS-7 cellswere transiently transfected with pHM6 containing full-length ratapoptosis-specific eIF-5A in the sense orientation. Expression of ratapoptosis-specific eIF-5A resulted in a 140% and 198% increase inphosphatidylserine exposure over control, in non-serum starved and serumstarved samples, respectively.

FIG. 31 illustrates enhanced apoptosis as reflected by increased nuclearfragmentation when COS-7 cells were transiently transfected with pHM6containing full-length rat apoptosis-specific eIF-5A in the senseorientation. Expression of rat apoptosis-specific eIF-5A resulted in a115% and 62% increase in nuclear fragmentation over control in untreatedand treated samples, respectively. FIG. 32 illustrates a comparison ofenhanced apoptosis under conditions in which COS-7 cells transientlytransfected with pHM6 containing full-length rat apoptosis-specificeIF-5A in the sense orientation were either given no further treatmentor treatment to induce apoptosis.

Example 4

The present example demonstrates modulation of apoptotic activityfollowing administration of apoptosis-specific eIF-5A.

COS-7 cells were either mock transfected, transfected with pHM6-LacZ ortransfected with pHM6-Sense rF5A (pHM6-Full length ratapoptosis-specific eIF-5A) and incubated for 40 hours. Five μg samplesof protein extract from each sample were fractionated by SDS-PAGE,transferred to a PVDF membrane, and Western blotted with a monoclonalantibody that recognizes Bcl-2. Rabbit anti-mouse IgG conjugated toperoxidase was used as a secondary antibody, and bound antibody wasdetected by chemiluminescence and exposure to x-ray film. Results areshown in FIG. 33. Less Bcl-2 is detectable in cells transfected withpHM6-Sense rF5A than in those transfected with pHM6-LacZ; thus showingthat Bcl-2 is down-regulated with the pHM6-sense rF5A construct.

Additional COS-7 cells were either mock transfected, transfected withpHM6-antisense 3′ rF5A. (pHM6-antisense 3′UTR of rat apoptosis-specificeIF-5A) or transfected with pHM6-Sense rF5A (pHM6-Full length ratapoptosis-specific eIF-5A). Forty hours after transfection, the cellswere induced to undergo apoptosis by withdrawal of serum for 48 hours.Five μg samples of protein extract from each sample were fractionated bySDS-PAGE, transferred to a PVDF membrane, and Western blotted with amonoclonal antibody that recognizes Bcl-2. Rabbit anti-mouse IgGconjugated to peroxidase was used as a secondary antibody, and boundantibody was detected by chemiluminescence and exposure to x-ray film.

Also additionally, COS-7 cells were either mock transfected, transfectedwith pHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-Full length ratapoptosis-specific eIF-5A) and incubated for 40 hours. Five μg samplesof protein extract from each sample were fractionated by SDS-PAGE,transferred to a PVDF membrane, and Western blotted with a monoclonalantibody that recognizes p53. Goat anti-mouse IgG conjugated to alkalinephosphatase was used as a secondary antibody, and bound antibody wasdetected a colorimetrically.

Finally, COS-7 cells were either mock transfected, transfected withpHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-Full length ratapoptosis-specific eIF-5A) and incubated for 40 hours. Five μg samplesof protein extract from each sample were fractionated by SDS-PAGE,transferred to a PVDF membrane, and probed with a monoclonal antibodythat recognizes p53. Corresponding protein blots were probed withanti-[HA]-peroxidase to determine the level of rat apoptosis-specificeIF-5A expression. Goat anti-mouse IgG conjugated to alkalinephosphatase was used as a secondary antibody, and bound antibody wasdetected by chemiluminescence.

FIG. 33 illustrates downregulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing frill-length ratapoptosis-specific eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. Less Bcl-2 is detectable incells transfected with pHM6-Sense rE5A than in those transfected withpHM6-LacZ.

FIG. 34 illustrates upregulation of Bcl-2 when COS-7 cells weretransiently transfected with pHM6 containing the 3′ end ofapoptosis-specific eIF-5A in the antisense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. More Bcl-2 is detectable incells transfected with pHM6-antisense 3′ rF5A than in those mocktransfected or transfected with pHM6-Sense rF5A.

FIG. 35 illustrates upregulation of c-Myc when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-specific eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. Higher levels of c-Myc isdetected in cells transfected with pHM6-Sense rF5A than in thosetransfected with pHM6-LacZ or the mock control.

FIG. 36 illustrates upregulation of p53 when COS-7 cells weretransiently transfected with pHM6 containing full-length ratapoptosis-specific eIF-5A in the sense orientation. The upper panelillustrates the Coomassie-blue-stained protein blot; the lower panelillustrates the corresponding Western blot. Higher levels of p53 isdetected in cells transfected with pHM6-Sense rF5A than in thosetransfected with pHM6-LacZ or the mock control.

FIG. 37 illustrates the dependence of p53 upregulation upon theexpression of pHM6-full length rat apoptosis-specific eIF-5A in COS-7cells. More rat apoptosis-specific eIF-5A is detectable in the firsttransfection than in the second transfection. In the Western blot probedwith anti-p53, the panel illustrates a correspondingCoomassie-blue-stained protein blot and the panel illustrates theWestern blot with p53. For the first transfection, more p53 isdetectable in cells transfected with pHM6-Sense rF5A than in thosetransfected with pHM6-LacZ or the mock control. For the secondtransfection in which there was less expression of ratapoptosis-specific eIF-5A, there was no detectable difference in levelsof p53 between cells transfected with pHM6-Sense rF5A, pHM6-LacZ or themock control.

Example 5

FIG. 47 depicts an experiment run on heart tissue to mimic the beatingof a human heart and the subsequent induced heart attack. FIG. 49 showsthe laboratory bench set up. A slice of human heart tissue removedduring valve replacement surgery was hooked up to electrodes. A smallweight as attached to the heart tissue to ease in measuring the strengthof the heart beats. The electrodes provided an electrical stimulus toget the tissue to start beating. The levels of gene expression for bothapoptosis-specific eIF-5A and proliferating eIF-5A were measured in theheart tissue before ischemia was induced. See FIG. 46. In thepre-ischemic heart tissue low levels both apoptosis-specific eIF-5A andproliferating eIF-5A were produced and their levels were in relativebalance. During this time, oxygen and carbon dioxide were delivered in abuffer to the heart at 92.5% and 7.5%, respectively. Later, the oxygenlevels was reduced and the nitrogen levels was increased, to induceischemia and finally a “heart attack.” The heart tissue stopped beating.The oxygen levels were then returned to normal, the heart tissue waspulsed again with an electrical stimulus to start the heart beatingagain. After the “heart attack” the expression levels ofapoptosis-specific eIF-5A and proliferating eIF-5A were again measured.This time, there was a significant increase in the level of expressionof the apoptosis-specific eIF-5A levels, whereas the increase in thelevel of expression of proliferating eIF-5A was noticeably less. SeeFIG. 46.

After the “heart attack” the heart did not beat as strong, as indicatedby less compression/movement of the attached weight, thus indicatingthat the heart tissue cells were being killed rapidly due to thepresence of apoptosis-specific eIF-5A.

The EKG results are depicted in FIG. 48. On the left side of the panelsa normal heart beat is shown (the pre-ischemic heart tissue). After the“heart attack” (straight line), and the re-initiation of the heart beat,the EKG shows decreased activity due to muscle cell death. The EKG showsrelative loss in strength of heart beat.

Example 6

The following examples provide cell culture conditions.

Human Lamina Cribrosa and Astrocyte Culture

Paired human eyes were obtained within 48 hours post mortem from the EyeBank of Canada, Ontario Division. Optic nerve heads (with attached pole)were removed and placed in Dulbecco's modified Eagle's medium (DIVIEM)supplemented with antibiotic/antimycotic, glutamine, and 10% FBS for 3hours. The optic nerve head (ONH) button was retrieved from each tissuesample and minced with fine dissecting scissors into four small pieces.Explants were cultured in 12.5 cm² plastic culture flasks in DMEMmedium. Growth was observed within one month in viable explants. Oncethe cells reached 90% confluence, they were trypsinized and subjected todifferential subculturing to produce lamina cribrosa (LC) and astrocytecell populations. Specifically, LC cells were subcultured in 25 cm²flasks in DMEM supplemented with gentamycin, glutamine, and 10% FBS,whereas astrocytes were expanded in 25 cm² flasks containing EBMcomplete medium (Clonetics) with no FBS. FBS was added to astrocytecultures following 10 days of subculture. Cells were maintained andsubcultured as per this protocol.

Cell populations obtained by differential subculturing werecharacterized for identity and population purity using differentialfluorescent antibody staining on 8 well culture slides. Cells were fixedin 10% formalin solution and washed three times with Dulbecco'sPhosphate Buffered Saline (DPBS). Following blocking with 2% nonfat milkin DPBS, antibodies were diluted in 1% BSA in DPBS and applied to thecells in 6 of the wells. The remaining two wells were treated with only1% bovine serum albumin (BSA) solution and no primary antibody ascontrols. Cells were incubated with the primary antibodies for one hourat room temperature and then washed three times with DPBS. Appropriatesecondary antibodies were diluted in 1% BSA in DPBS, added to each welland incubated for 1 hour. Following washing with DPBS, the chambersseparating the wells of the culture slide were removed from the slide,and the slide was immersed in double distilled water and then allowed toair-dry. Fluoromount (Vector Laboratories) was applied to each slide andoverlayed by 22×60 mm coverglass slips.

Immunofluorescent staining was viewed under a fluorescent microscopewith appropriate filters and compared to the control wells that were nottreated with primary antibody. All primary antibodies were obtained fromSigma unless otherwise stated. All secondary antibodies were purchasedfrom Molecular Probes. Primary antibodies used to identify LC cellswere: anti-collagen I, anti-collagen IV, anti-laminin, anti-cellularfibronectin. Primary antibodies used to identify astrocytes were:anti-galactocerebroside (Chemicon International), anti-A2B5 (ChemiconInternational), anti-NCAM, anti-human Von willebrand Factor. Additionalantibodies used for both cell populations included anti-glial fibrillary(GFAP) and anti-alpha-smooth muscle actin. Cell populations weredetermined to be comprised of LC cells if they stained positively forcollagen I, collagen IV, laminin, cellular fibronectin, alpha smoothmuscle actin and negatively for glial fibrillary (GFAP). Cellpopulations were determined to be comprised of astrocytes if theystained positively for NCAM, glial fibrillary (GFAP), and negatively forgalactocerebroside, A2B5, human Von willebrand Factor, and alpha smoothmuscle actin.

In this preliminary study, three sets of human eyes were used toinitiate cultures. LC cell lines #506, #517, and #524 were establishedfrom the optic nerve heads of and 83-Year old male, a 17-year old male,and a 26-year old female, respectively. All LC cell lines have beenfully characterized and found to contain greater than 90% LC cells.

RKO Cell Culture

RKO (American Type Culture Collection CRL-2577), a human colon carcinomacell line expressing wild-type p53, was used to test the antisenseoligonucleotides for the ability to suppress apoptosis-specific eIF-5Aprotein expression. RKO were cultured in Minimum Essential Medium Eagle(MEM) with non-essential amino acids, Earle's salts, and L-glutamine.The culture media was supplemented with 10% fetal bovine serum (FBS) and100 units of penicillin/streptomycin. The cells were grown at 37° C. ina humidified environment of 5% CO₂ and 95% air. The cells weresubcultured every 3 to 4 days by detaching the adherent cells with asolution of 0.25% trypsin and 1 mM EDTA. The detached cells weredispensed at a split ratio of 1:10 to 1:1.2 into a new culture dish withfresh media.

HepG2 Cell Culture

HepG2, a human hepatocellular carcinoma cell line, was used to test theability of an antisense oligo directed against human apoptosis-specificeIF-5A to block production of TNF-α in response to treatment with IL-1β.HepG2 cells were cultured in DMEM supplemented with gentamycin,glutamine, and 10% FBS and grown at 37° C. in a humidified environmentof 5% CO₂ and 95% air.

Example 7 Induction of Apoptosis

Apoptosis was induced in RKO and lamina cribrosa cells using ActinomycinD, an RNA polymerase inhibitor, and camptothecin, a topoisomeraseinhibitor, respectively. Actinomycin D was used at a concentration of0.25 μg/ml and camptothecin was used at a concentration of 20, 40, or 50μM. Apoptosis was also induced in lamina cribrosa cells using acombination of camptothecin (50 μM) and TNF-α (10 ng/ml). Thecombination of camptothecin and TNF-α was found to be more effective atinducing apoptosis than either camptothecin or TNF-α alone.

Antisense Oligonucleotides

A set of three antisense oligonucleotides targeted against humanapoptosis-specific eIF-5A were designed by, and purchased from, MoleculaResearch Labs. The sequence of the first antisense oligonucleotidetargeted against human apoptosis-specific eIF-5A (#1) was 5′ CCT OTC TCGAAG TCC AAG TC 3′ (SEQ ID NO: 63). The sequence of the second antisenseoligonucleotide targeted against human apoptosis-specific eIF-5A (#2)was 5′ GGA CCT TGG CGT GOC CGT GC 3° (SEQ ID NO: 64). The sequence ofthe third antisense oligonucleotide targeted against humanapoptosis-specific eIF-5A (#3) was 5′ CTC GTA CCT CCC CGC TCT CC 3′ (SEQID NO: 65). The control oligonucleotide had the sequence 5′ CGT ACC GOTACG OTT CCA GG 3′ (SEQ ID NO: 66). A fluorescein isothiocyanate(FITC)-labeled antisense oligonucleotide (Molecula Research Labs) wasused to monitor transfection efficiency and had the sequence 5′ GGA CCTTGG CGT GGG CGT GCX 3′ (SEQ ID NO: 67),

where X is the FITC label. All antisense oligonucleotides were hillyphosphorothioated.

Transfection of Antisense Oligonucleotides

The ability of the apoptosis-specific eIF-5A antisense oligonucleotidesto block apoptosis-specific eIF-5A protein expression was tested in RKOcells. RKO cells were transfected with antisense oligonucleotides usingthe transfection reagent, Oligofectamine (Invitrogen). Twenty four hoursprior to transfection, the cells were split onto a 24 well plate at157,000 per well in MEM media supplemented with 10% FBS but lackingpenicillin/streptomycin. Twenty four hours later the cells had generallyreached a confluency of approximately 50%. RKO cells were either mocktransfected, or transfected with 100 nM or 200 nM of antisenseoligonucleotide. Transfection medium sufficient for one well of a 24well plate was prepared by diluting 0, 1.25, or 2.5 μl of a 20 μM stockof antisense oligonucleotide with serum-free MEM to a final volume of42.5 and incubating the mixture at room temperature for 15 minutes. 1.5μl of Oligofectamine was diluted in 6 μl of serum-free MEM and incubatedfor 7.5 minutes at room temperature. After 5 minutes the dilutedOligofectamine mixture was added to the DNA mixture and incubatedtogether at room temperature for 20 minutes. The cells were washed oncewith serum-free MEM before adding 200 μl of MEM to the cells andoverlaying 50 μl of transfection medium. The cells were placed back inthe growth chamber for 4 hours. After the incubation, 125 μl of MEM+30%FBS was added to the cells. The cells were then cultured for a further48 hours, treated with 0.25 μg/ml Actinomycin D for 24 hours, and thencell extract was harvested for Western blot analysis.

Transfection of lamina cribrosa cells was also tested using 100 and 200nM antisense oligonucleotide and Oligofectamine using the same proceduredescribed for RKO cells. However, effective transfection of laminacribrosa cells was achieved by simply adding antisense oligonucleotide,diluted from 1 μM to 10 μM in serum-free media, to the cells for 24hours and thereafter replacing the media with fresh antisenseoligonucleotides diluted in serum-containing media every 24 hours for atotal of two to five days.

The efficiency of antisense oligonucleotide transfection was optimizedand monitored by performing transfections with an FITC-labeled antisenseoligonucleotide having the same sequence as apoptosis-specific eIF-5Aantisense oligonucleotide #2 (SEQ ID NO:64) but conjugated to FITC atthe 3′ end. RKO and lamina cribrosa cells were transfected with theFITC-labeled antisense oligonucleotide on an 8-well culture slide.Forty-eight hours later the cells were washed with PBS and fixed for 10minutes in formaldehyde in PBS. The wells were removed and mountingmedia (Vectashield) was added, followed by a coverslip. The cells werethen visualized under UV light on a fluorescent microscope nucleus usinga fluorescein filter (Green H546, filter set 48915) and cellsfluorescing bright green were determined to have taken up theoligonucleotide.

Detection of Apoptosis

Following transfection of lamina cribosa cells with antisenseoligonucleotides and induction of apoptosis with camptothecin, thepercentage of cells undergoing apoptosis in cells treated with eithercontrol antisense oligonucleotide or antisense oligonucleotideapoptosis-specific eIF-5A SEQ ID NO:26 was determined. Two methods wereused to detect apoptotic lamina cribosa cells—Hoescht staining andDeadEnd™ Fluorometric TUNEL. The nuclear stain, Hoescht, was used tolabel the nuclei of lamina cribosa cells in order to identify apoptoticcells based on morphological features such as nuclear fragmentation andcondensation. A fixative, consisting of a 3:1 mixture of absolutemethanol and glacial acetic acid, was prepared immediately before use.An equal volume of fixative was added to the media of cells growing on aculture slide and incubated for 2 minutes. The media/fixative mixturewas removed from the cells and discarded and 1 ml of fixative was addedto the cells. After 5 minutes the fixative was discarded and 1 ml offresh fixative was added to the cells and incubated for 5 minutes. Thefixative was discarded and the cells were air-dried for 4 minutes beforeadding 1 ml of Hoescht stain (0.5 μg/ml Hoescht 33258 in PBS). After a10 minute incubation in the dark, the staining solution was discarded,the chambers separating the wells of the culture slide were removed, andthe slide was washed 3 times for 1 minute with deionized water. Afterwashing, a few drops of Mcllvaine's buffer (0.021 M citric acid, 0.058 MNa₂HPO₄.7H₂O; pH 5.6) was added to the cells and overlaid with acoverslip. The stained cells were viewed under a fluorescent microscopeusing a UV filter. Cells with brightly stained or fragmented nuclei werescored as apoptotic. A minimum of 200 cells were counted per well.

The DeadEnd™ Fluorometric TUNEL, (Promega) was used to detect the DNAfragmentation that is a characteristic feature of apoptotic cells.Following Hoescht staining, the culture slide was washed briefly withdistilled water, and further washed by immersing the slide twice for 5minutes in PBS (137 mM NaCl, 2.68 mM Ka, 1.47 mM KH₂PO₄, 8.1mM.Na₂HPO₄), blotting the slide on paper towel between washes. The cellswere permeabilized by immersing them in 0.2% Triton X-100 in PBS for 5minutes. The cells were then washed again by immersing the slide twicefor 5 minutes in PBS and blotting the slide on paper towel betweenwashes. 25 μl of equilibration buffer [200 mM potassium cacodylate (pH6.6), 25 mM Tris-HCl (pH 6.6), 0.2 mM dithiothreitol, 0.25 mg/ml bovineserum albumin, and 2.5 mM cobalt chloride] was added per well andincubated for 5 to 10 minutes. During equilibration, 30 μl of reactionmixture was prepared for each well by mixing in a ratio of 45:5:1,respectively, equilibration buffer, nucleotide mix [50 μMfluorescein-12-dUTP, 100 μM dATP, 10 mM Tris-HCl (pH 7.6), and 1 mMEDTA], and terminal deoxynucleotidyl transferase enzyme (Tdt, 25 U/μl).After the incubation in equilibration buffer, 30 μl of reaction mixturewas added per well and overlayed with a coverslip. The reaction wasallowed to proceed in the dark at 37° C. for 1 hour. The reaction wasterminated by immersing the slide in 2×SSC [0.3 M NaCl, and 30 mM sodiumcitrate (pH 7.0)] and incubating for 15 minutes. The slide was thenwashed by immersion in PBS three times for 5 minutes. The PBS wasremoved by sponging around the wells with a Kim wipe, a drop of mountingmedia (Oncogene research project, JA1750-4 ML) was added to each well,and the slide was overlayed with a coverslip. The cells were viewedunder a fluorescent microscope using a UV filter (UV-G 365, filter set487902) in order to count the Hoescht-stained nuclei. Any cells withbrightly stained or fragmented nuclei were scored as apoptotic. Usingthe same field of view, the cells were then viewed using a fluoresceinfilter (Green H546, filter set 48915) and any nuclei fluorescing brightgreen were scored as apoptotic. The percentage of apoptotic cells in thefield of view was calculated by dividing the number of bright greennuclei counted using the fluorescein filter by the total number ofnuclei counted under the UV filter. A minimum of 200 cells were countedper well.

FIGS. 54-57 depict the results of these studies. The percentage ofapoptotic cells in samples having been transfected withapoptosis-specific eIF-5A is clearly much less than seen in cells havingbeen transfected with the control oligonucleotide.

Protein Extraction and Western Blotting

Protein from transfected RKO cells was harvested for Western blotanalysis by washing the cells with PBS, adding 40 μl of hot lysis buffer[0.5% SDS, 1 mM dithiothreitol, 50 mM Tris-HCl (pH 8.0)] per well. Thecells were scraped and the resulting extract was transferred to amicrofuge tube, boiled for 5 minutes, and stored at −20° C. The proteinwas quantitated using the Bio-Rad Protein Assay (Bio-Rad) according tothe manufacturer's instructions.

For Western blotting 5 μg of total protein was separated on a 12%SDS-polyacrylamide gel. The separated proteins were transferred to apolyvinylidene difluoride membrane. The membrane was then incubated throne hour in blocking solution (5% skim milk powder in PBS) and washedthree times for 15 minutes in 0.05% Tween-20/PBS. The membrane wasstored overnight in PBS-T at 4° C. After being warmed to roomtemperature the next day, the membrane was blocked for 30 seconds in 1μg/ml polyvinyl alcohol. The membrane was rinsed 5 times in deionizedwater and then blocked for 30 minutes in a solution of 5% milk in 0.025%Tween-20/PBS. The primary antibody was preincubated for 30 minutes in asolution of 5% milk in 0.025% Tween-20/PBS prior to incubation with themembrane.

Several primary antibodies were used. A monoclonal antibody fromOncogene which recognizes p53 (Ab-6) and a polyclonal antibody directedagainst a synthetic peptide (amino-CRLPEGDLGKEIEQKYD-carboxy) (SEQ IDNO: 68) homologous to the c-terminal end of human apoptosis-specificeIF-5A that was raised in chickens (Gallus Immunotech). An anti-β-actinantibody (Oncogene) was also used to demonstrate equal loading ofprotein. The monoclonal antibody to p53 was used at a dilution of 0.05μg/ml, the antibody against apoptosis-specific eIF-5A was used at adilution of 1:1000, and the antibody against actin was used at adilution of 1:20,000. After incubation with primary antibody for 60 to90 minutes, the membrane was washed 3 times for 15 minutes in 0.05%Tween-20′PBS. Secondary antibody was then diluted in 1% milk in 0.025%Tween-20/PBS and incubated with the membrane for 60 to 90 minutes. Whenp53 (Ab-6) was used as the primary antibody, the secondary antibody usedwas a rabbit anti-mouse IgG conjugated to peroxidase (Sigma) at adilution of 1:5000. When anti-apoptosis-specific eIF-5A was used as theprimary antibody, a rabbit anti-chicken IgY conjugated to peroxidase(Gallus Immunotech) was used at a dilution of 1:5000. The secondaryantibody used with actin was a goat anti-mouse IgM conjugated toperoxidase (Calbiochem) used at a dilution of 1:5000. After incubationwith the secondary antibody, the membrane was washed 3 times in PBS-T.

The ECL Plus Western blotting detection kit (Amersham Pharmacia Biotech)was used to detect peroxidase-conjugated bound antibodies. In brief, themembrane was lightly blotted dry and then incubated in the dark with a40:1 mix of reagent A and reagent B for 5 minutes. The membrane wasblotted dry, placed between sheets of acetate, and exposed to X-ray filmfor time periods varying from 10 seconds to 30 minutes. The membrane wasstripped by submerging the membrane in stripping buffer [100 mM2-Mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7)], andincubating at 50° C. for 30 minutes. The membrane was then rinsed indeionized water and washed twice for 10 minutes in large volumes of0.05% Tween-20/PBS. Membranes were stripped and re-blotted up to threetimes.

Example 8 Construction of siRNA

Small inhibitory RNAs (siRNAs) directed against human apoptosis-specificeIF-5A were used to specifically suppress expression ofapoptosis-specific eIF-5A in MO and lamina cribrosa cells. Six siRNAswere generated by in vitro transcription using the Silencer™ siRNAConstruction Kit (Ambion Inc.). Four siRNAs were generated against humanapoptosis-specific eIF-5A (siRNAs #1 to #4) (SEQ NO:30-33). Two siRNAswere used as controls; an siRNA directed against GAPDH provided in thekit, and an siRNA (siRNA #5) (SEQ ID NO: 34) which had the reversesequence of the apoptosis-specific eIF-5A siRNA #1 (SEQ ID NO:30) butdoes not itself target apoptosis-specific eIF-5A. The siRNAs weregenerated according to the manufacturer's protocol. In brief, DNAoligonucleotides encoding the desired siRNA strands were used astemplates for T7 RNA polymerase to generate individual strands of thesiRNA following annealing of a T7 promoter primer and a fill-in reactionwith Klenow fragment. Following transcription reactions for both thesense and antisense strands, the reactions were combined and the twosiRNA strands were annealed, treated with DNase and RNase, and thencolumn purified. The sequence of the DNA oligonucleotides (T7 primerannealing site underlined) used to generate the siRNAs were: siRNA #1antisense 5′ AAAGGAATGACTTCCAGCTGACCTGTCTC 3′ (SEQ ID NO:69) and siRNA#1 sense 5′ AATCAGCTGGAAGTCATTCCTCCTGTCTC 3′ (SEQ ID NO:70); siRNA #2antisense 5′ AAGATCGTCGAGAIGTCTACTCCTGTCTC 3′ (SEQ ID NO:71) and siRNA#2 sense 5′ AAAGTAGACATCTCGACGATCCCTGTCTC 3′ (SEQ ID NO:72); siRNA. #3antisense 5′ AAGGTCCATCTGGTTGGTATTCCTGTCTC 3′ (SEQ ID NO:73) and siRNA#3 sense 5′ AAAATACCAACCAGATGGACCCCTGTCTC 3′ (SEQ ID NO:74) siRNA #4antisense 5′ AAGCTGGACTCCTCCTACACACCTGTCTC 3′ (SEQ ED NO:75) and siRNA#4 sense 5′ AATGTGTAGGAGGAGTCCAGCCCTGTCTC 3′ (SEQ ID NO:76); siRNA 5antisense 5′ AAAGTCGACCTICAGTAAGGACCTGFCTC 3′ (SEQ ID NO:77) and siRNA#5 sense 5′ AATCCTTACTGAAGGTCGACTCCTGTCTC 3′ (SEQ ID NO:78).

The Silencer™ siRNA Labeling Kit—FAM (Ambion) was used to label GAPDHsiRNA with FAM in order to monitor the uptake of siRNA into RKO andlamina cribrosa cells. After transfection on 8-well culture slides,cells were washed with PBS and fixed for 10 minutes in 3.7% formaldehydein PBS. The wells were removed and mounting media (Vectashield) wasadded, followed by a coverslip. Uptake of the FAM-labeled siRNA wasvisualized under a fluorescent microscope under UV light using afluorescein filter. The GAPDH siRNA was labeled according to themanufacturer's protocol.

Transfection of siRNA

RKO cells and lamina cribrosa cells were transfected with siRNA usingthe same transfection protocol. RKO cells were seeded the day beforetransfection onto 8-well culture slides or 24-well plates at a densityof 46,000 and 105,800 cells per well, respectively. Lamina cribrosacells were transfected when cell confluence was at 40 to 70% and weregenerally seeded onto 8-well culture slides at 7500 to 10,000 cells perwell three days prior to transfection. Transfection medium sufficientfor one well of an 8-well culture slide was prepared by diluting 25.5pmoles of siRNA stock to a final volume of 21.2 μl in Opti-Mem (Sigma).0.425 μl of Lipofectamine 2000 was diluted to a final volume of 21.2 μlin Opti-Mem and incubated for 7 to 10 minutes at room temperature. Thediluted Lipofectamine 2000 mixture was then added to the diluted siRNAmixture and incubated together at room temperature for 20 to 30 minutes.The cells were washed once with serum-free media before adding 135 μl ofserum-free media to the cells and overlaying the 42.4 μl of transfectionmedium. The cells were placed back in the growth chamber for 4 hours.After the incubation, 65 μl it of serum-free media +30% FBS was added tothe cells. Transfection of siRNA into cells to be used for Western blotanalysis were performed in 24-well plates using the same conditions asthe transfections in 8-well slides except that the volumes wereincreased by 2.3 fold.

Following transfection, RKO and lamina cribrosa cells were incubated for72 hours prior to collection of cellular extract for Western blotanalysis. In order to determine the effectiveness of the siRNAs directedagainst apoptosis-specific eIF-5A to block apoptosis, lamina cribrosacells were treated with 50 μM of camptothecin (Sigma) and 10 ng/ml ofTNF-α (Leinco Technologies) to induce apoptosis either 48 or 72 hoursafter transfection. The cells were stained with Hoescht either 24 or 48hours later in order to determine the percentage of cells undergoingapoptosis.

Example 9 Quantification of HepG2 TNF-α Production

HepG2 cells were plated at 20,000 cells per well onto 48-well plates.Seventy two hours later the media was removed and fresh media containingeither 2.5 μM control antisense oligonucleotide or 2.5 μM antisenseoligonucleotide apoptosis-specific eIF-5A #2 was added to the cells.Fresh media containing antisense oligonucleotides was added after twentyfour hours. After a total of 48 hours incubation with theoligonucleotides, the media was replaced with media containinginterleukin 1β (IL-1β, 1000 pg/ml; Leinco Technologies) and incubatedfor 6 hours. The media was collected and frozen (20° C.) for TNF-αquantification. Additional parallel incubations with untreated cells(without antisense oligonucleotide and IL-1β) and cells treated withonly IL-1β were used for controls. All treatments were done induplicate. TNF-α released into the media was measured by ELISA assays(Assay Designs Inc.) according to the manufacturer's protocol.

Example 10

The following experiments show that antisense apoptosis-specific eIF-5Anucleotides were able to inhibit expression of apoptosis-specific eIF-5Aas well as p53.

RKO cells were either left untransfected, mock transfected, ortransfected with 200 nM of antisense oligonucleotides apoptosis-specificeIF-5A #1, #2, or #3 (SEQ ID NO: 25, 26, and 27). RKO cells were alsotransfected with 100 nM of antisense oligonucleotide apoptosis-specificeIF-5A #2 (SEQ ID NO:26). Forty-eight hours after transfection, thecells were treated with 0.25 Actinomycin D. Twenty-four hours later, thecell extract was harvested and 5 μg of protein from each sample wasseparated on an SDS-PAGE gel, transferred to a PVDF membrane, andWestern blotted with an antibody against apoptosis-specific eIF-5A.After chemiluminescent detection, the membrane was stripped and reprobedwith an antibody against p53. After chemiluminescent detection, themembrane was stripped again and reprobed with an antibody against actin.FIG. 52 which shows the levels of protein produced by RKO cells afterbeing treated with antisense oligo 1, 2 and 3 (to apoptosis-specificeIF-5a) (SEQ ID NO:25, 26, and 27, respectively). The RKO cells producedless apoptosis-specific eIF-5A as well as less p53 after having beentransfected with the antisense apoptosis-specific eIF-5A nucleotides.

Example 11

The following experiments show that apoptosis-specific eIF-5Anucleotides were able to reduce apoptosis.

In one experiment, the lamina cribrosa cell line #506 was either (A)transfected with 100 nM of FITC-labeled antisense oligonucleotide usingOligofectamine transfection reagent or (B) transfected with 10 μM ofnaked FITC-labeled antisense oligonucleotide diluted directly inserum-free media. After 24 hours fresh media containing 10% FBS andfresh antisense oligonucleotide diluted to 10 μM was added to the cells.The cells, (A) and (B), were fixed after a total of 48 hours andvisualized on a fluorescent microscope under UV light using afluorescein filter. FIG. 53 shows uptake of the fluorescently labeledantisense oligonucleotide.

In another experiment, the lamina cribrosa cell line #506 wastransfected with 10 μM of either the control antisense oligonucleotideor antisense oligonucleotide apoptosis-specific eIF-5A #2 (SEQ ID NO:26)for a total of 4 days. Forty-eight, hours after beginning antisenseoligonucleotide treatment, the cells were treated with either 20 μM: or40 μM camptothecin for 48 hours. Antisense oligonucleotide andcamptothecin-containing media was changed daily. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht andTUNEL. See FIG. 54.

In another experiment, the lamina cribrosa cell line #506 wastransfected with 10 μM of either the control antisense oligonucleotideor antisense oligonucleotide apoptosis-specific eIF-5A #2 (SEQ IDNO:26). Twenty-four hours later the media was changed and freshantisense oligonucleotides were added. Forty-eight hours after beginningantisense oligonucleotide treatment, the antisense-oligonucleotides wereremoved and the cells were treated with 20 μM camptothecin for 3 days.The camptothecin-containing media was changed daily. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht andTUNEL. See FIG. 55.

In yet another experiment, the lamina cribrosa cell line #517 wastransfected with 1 μM of either the control antisense oligonucleotide orantisense oligonucleotide apoptosis-specific eIF-5A #2 (SEQ ID NO:26)for a total of five days. Forty-eight hours after beginning antisenseoligonucleotide treatment, the cells were treated with 20 μMcamptothecin thr either 3 or 4 days. Antisense oligonucleotide andcamptothecin-containing media was changed daily. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht andTUNEL. See FIG. 56.

In another experiment, the lamina cribrosa cell line #517 wastransfected with 2.5 μM of either the control antisense oligonucleotideor antisense oligonucleotide apoptosis-specific eIF-5A #2 (SEQ ED NO:26)for a total of five days. Forty-eight hours after beginning antisenseoligonucleotide treatment, the cells were treated with 40 μMcamptothecin for 3 days, Antisense oligonucleotide andcamptothecin-containing media was changed daily. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht. SeeFIG. 57.

In another experiment, the lamina cribrosa cell line #517 wastransfected with either 1 μM or 2.5 μM of either the control antisenseoligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A#2 (SEQ ID NO:26) for a total of five days. Forty-eight hours afterbeginning antisense oligonucleotide treatment, the cells were treatedwith 40 μM camptothecin for 3 days. Antisense oligonucleotide andcamptothecin-containing media was changed daily. The percentage ofapoptotic cells was determined by labeling the cells with Hoescht. SeeFIG. 58.

In another experiment, the lamina cribrosa cell line #517 was lefteither untreated, or was treated with 10 ng/ml TNF-α, 50 μMcamptothecin, or 10 ng/ml TNF-α and 50 μM camptothecin. The percentageof apoptotic cells was determined by labeling the cells with Hoescht.See FIG. 59.

In another experiment, the lamina cribrosa cell lines #506 and #517 weretransfected with either 2.5 μM or 5 μM of either the control antisenseoligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A#2 (SEQ ID NO:26) for a total of two days. Fresh media containingantisense oligonucleotides was added after 24 hours. Forty-eight hoursafter beginning antisense oligonucleotide treatment, the cells weretreated with 50 μM camptothecin and 10 ng/ml TNF-α for 2 days. Thepercentage of apoptotic cells was determined by labeling the cells withHoescht. See FIG. 60.

In another experiment, the lamina cribrosa cell lines #506, #517, and#524 were transfected with 2.5 μM of either the control antisenseoligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A#2 (SEQ ID NO:26) for a total of two days. Fresh media containingantisense oligonucleotides was added after 24 hours. Forty-eight hoursafter beginning antisense oligonucleotide treatment, the cells weretreated with 50 μM camptothecin and 10 ng/ml TNF-α for 2 days. Thepercentage of apoptotic cells was determined by labeling the cells withHoescht. See FIG. 61.

Example 12

The following experiments show that cells transfected with siRNAstargeted against apoptosis-specific eIF-5A expressed lessapoptosis-specific eIF-5A. The experiments also show that siRNAstargeted against apoptosis-specific eIF-5A were able to reduceapoptosis.

In one experiment, the lamina cribrosa cell line #517 was transfectedwith 100 nM of FAM-labeled siRNA using Lipofectamine 2000 transfectionreagent either with serum (A) or without serum (B) during transfection.The cells. (A) and (B), were fixed after a total of 24 hours andvisualized on a fluorescent microscope under UV light using afluorescein filter. See FIG. 62.

In another experiment., RKO cells were transfected with 100 nM of siRNAeither in the presence or absence of serum during the transfection. SixsiRNAs were transfected, two control siRNAs (siRNA #5 (SEQ ID NO:34) andone targeted against GAPDH) and four targeted against apoptosis-specificeIF-5A (siRNA #1 to #4) (SEQ ID NO:30-33). Seventy-two hours aftertransfection, the cell extract was harvested and 5 μg of protein fromeach sample was separated on an SDS-PAGE gel, transferred to a PVDFmembrane, and Western blotted with an antibody againstapoptosis-specific eIF-5A. After chemiluminescent detection, themembrane was stripped and re-probed with an antibody against bcl-2.After chemiluminescent detection, the membrane was stripped again andre-probed with an antibody against actin. See FIG. 63.

In another experiment, lamina Cribrosa cell lines #506 and #517 weretransfected with 100 nM of siRNA. Six siRNAs were transfected, twocontrol siRNAs (siRNA #5 (SEQ ID NO:34) and one targeted against GAPDH)and four targeted against apoptosis-specific eIF-5A (siRNA #1 to #4)(SEQ ID NO:30-33). Seventy-two hours after transfection, the cellextract was harvested and 5 μg of protein from each sample was separatedon an SDS-PAGE gel, transferred to a PVDF membrane, and Western blottedwith an antibody against apoptosis-specific eIF-5A. Afterchemiluminescent detection, the membrane was stripped and re-probed withan antibody against actin. See FIG. 64.

In another experiment, the lamina cribrosa cell #506 was transfectedwith 100 nm of siRNA. Six siRNAs were transfected, two control siRNAs(siRMA. #5 (SEQ ID NO:34) and one targeted against GAPDH) and fourtargeted against apoptosis-specific eIF-5A (siRNA #1 to #4) (SEQ IDNO:30-33). Forty-eight hours after transfection, the media was replacedwith media containing 50 μM camptothecin and 10 ng/ml TNF-α. Twenty-fourhours later, the percentage of apoptotic cells was determined bylabeling the cells with Hoescht. See FIG. 65

In another experiment, the lamina cribrosa cell line #506 wastransfected with 100 nm of siRNA. Six siRNAs were transfected, twocontrol siRNAs (siRNA #5 (SEQ ID NO:34) and one targeted against GAPDH)and four targeted against apoptosis-specific eIF-5A (siRNA #1 to #4)(SEQ ID NO:30-33). Seventy-two hours after transfection, the media wasreplaced with media containing 50 μM camptothecin and 10 ng/ml TNF-α.Twenty-four hours later, the percentage of apoptotic cells wasdetermined by labeling the cells with Hoescht. See FIG. 66.

In another experiment, the lamina cribrosa cell line #506 was eitherleft untransfected or was transfected with 100 nm of siRNA. Six siRNAswere transfected, two control siRNAs (siRNA #5 (SEQ ID NO:34) and onetargeted against GAPDH) and four targeted against apoptosis-specificeIF-5A (siRNA #1 to #4) (SEQ ID NO:30-33). Seventy-two hours aftertransfection, the media was replaced with media containing 50 μMcamptothecin and 10 ng/ml TNF-α. Fresh media was also added to theuntransfected, untreated control cells. Forty-eight hours later, thepercentage of apoptotic cells was determined by labeling the cells withHoescht. See FIG. 67.

Photographs of Hoescht-stained lamina cribrosa cell line #506transfected with siRNA and treated with camptothecin and TNF-α from theexperiment described in FIG. 67 and example 13 are provided in FIG. 68.

Example 13

This example shows that treating a human cell line with antisenseoligonucleotides directed against apoptosis-specific eIF-5A causes thecells to produce less TNF-α.

HepG2 cells were treated with 2.5 μM of either the control antisenseoligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A#2 for a total of two days. Fresh media containing antisenseoligonucleotides was added after 24 hours. Additional cells were leftuntreated for two days. Forty-eight hours after the beginning oftreatment, the cells were treated with IL-1β (1000 pg/ml) in fresh mediafor 6 hours. At the end of the experiment, the media was collected andfrozen (−20° C.) for TNF-α quantification. TNF-α released into the mediawas measured using ELISA assays purchased from Assay Designs Inc. SeeFIG. 69. Cells that were transfected with antisense oligonucleotides ofapoptosis-specific eIF-5A produced less TNF-α.

Example 14

HT-29 cells (human colon adenocarcinoma) were transfected with either ansiRNA against apoptosis-specific eIF-5A or with a control siRNA with thereverse sequence. The siRNA used is as follows:

Position 690 (3′UTR) % G/C = 48 5′ AAGCUGGACUCCUCCUACACA 3′(SEQ ID NO: 79)The control siRNA used is as follows:

% G/C = 39 5′ AAACACAUCCUCCUCAGGUCG 3′ (SEQ ID NO: 80)After 48 hours the cells were treated with interferon-gamma (IFN-gamma)for 16 hours. After 16 hours the cells were washed with fresh media andtreated with lipopolysaccharide (LPS) for 8 or 24 hours. At each timepoint (8 or 24 hours) the cell culture media was removed from the cells,frozen, and the TNF-alpha present in the media was quantitated by ELISA.The cell lysate was also harvested, quantitated for protein, and used toadjust the TNF-alpha values to pg/mg protein (to adjust for differencesin cell number in different wells). The results of the Western blot andElisa are provided in FIGS. 74 A and B. FIG. 75 are the results of thesame experiment except the cells were at a higher density.

Example 15 Tissue Culture Conditions of U-937 Cell Line

U-937 is a human monocyte cell line that grows in suspension and willbecome adherent and differentiate into macrophages upon stimulation withPMA (ATCC Number CRL-1593.2) (cells not obtained directly from ATCC).Cells were maintained in RPMI 1640 media with 2 mM L-glutamine, 1.5 g/L,sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvateand 10% fetal bovine serum in a 37° C. CO₂ (5%) incubator. Cells weresplit into fresh media (1:4 or 1:5 split ratio) twice a week and thecell density was always kept between 105 and 2×106 cells/ml. Cells werecultured in suspension in tissue culture-treated plastic T-25 flasks andexperiments were conducted in 24-well plates.

Time Course Experiment

Two days before the start of an experiment, the cell density wasadjusted to 3×105 cells/ml media. On the day of the experiment, thecells were harvested in log phase. The cell suspension was transferredto 15 ml tubes and centrifuged at 400×g for 10 mins at room temperature.The supernatant was aspirated and the cell pellet was washed/resuspendedwith fresh media. The cells were again centrifuged at 400×g for 10 mins,the supernatant was aspirated, and the cell pellet was finallyresuspended in fresh Media. Equal volumes of cell suspension and trypanblue solution (0.4% trypan blue dye in PBS) were mixed and the livecells were counted using a haemocytometer and a microscope. The cellswere diluted to 4×105 cells/ml.

A 24-well plate was prepared by adding either PMA or DMSO (vehiclecontrol) to each well. 1 ml of cell suspension was added to each well sothat each well contained 400,000 cells, 0.1% DMSO+/−162 nM PMA. Thecells were maintained in a 37° C. CO₂ (5%) incubator. Separate wells ofcells were harvested at times 0, 24, 48, 72, 96, 99 and 102 h. See FIG.76 for a summary of the experimental time points and additions.

The media was changed at 72 h. Since some cells were adherent and otherswere in suspension, care was taken to avoid disrupting the adherentcells. The media from each well was carefully transferred intocorresponding microcentrifuge tubes and the tubes were centrifuged at14,000×g for 3 mm. The tubes were aspirated, the cell pellets wereresuspended in fresh media (1 ml, (−) DMSO, (−) PMA), and returned totheir original wells. The cells become quiescent in this fresh mediawithout PMA. At 96 h, LPS (100 ng/ml) was added and cells were harvestedat 3 h (99 h) and 6 h (102 h) later.

At the time points, the suspension cells and media were transferred fromeach well into microcentrifuge tubes. The cells were pelleted at14,000×g for 3 min. The media (supernatant) was transferred to cleantubes and stored (−20° C.) for ELISA/cytokine analysis. The cellsremaining in the wells were washed with PBS (1 ml, 37° C.) and this PBSwas also used to wash the cell pellets in the correspondingmicrocentrifuge tubes. The cells were pelleted again at 14,000×g for 3min. The cells were lysed with boiling lysis buffer (50 mM Tris pH 7.4and 2% SDS). The adherent cells and the suspension cells from each wellwere pooled. The samples were boiled and then stored at −20° C.

Western Blotting

The protein concentration in each cell sample was determined by the BCA(bicinchoninic acid) method using BSA (bovine serum albumin) as thestandard protein. Protein samples (5 μg total protein) were separated by12% SDS-PAGE electrophoresis and transferred to PVDF membranes. Themembranes were blocked with polyvinyl alcohol (1 μg/ml, 30 sec) and with5% skim milk in PBS-t (1 h). The membranes were probed with a mousemonoclonal antibody raised against human eIF-5A (BD Biosciences cat#611976; 1:20,000 in 5% skim milk, 1 h). The membranes were washed 3×10mins PBS-t. The secondary antibody was a horseradishperoxidase-conjugated antimouse antibody (Sigma, 1:5000 in 1% skim milk,1 h). The membranes were washed 3×10 mins PBS-t. The protein bands werevisualized by chemiluminescence (ECL detection system, AmershamPharmacia Biotech).

To demonstrate that similar amounts of protein were loaded on each gellane, the membranes were stripped and reprobed for actin. Membranes werestripped (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7; 50°C. for 30 mins), washed, and then blocked as above. The membranes wereprobed with actin primary antibody (actin monoclonal antibody made inmouse; Oncogene, Ab-1; 1:20,000 in 5% skim milk). The secondaryantibody, washing, and detection were the same as above.

FIG. 77 shows that eIF5A is upregulated during monocyte (U-397)differentiation and subsequent TNF-α secretion.

Example 16 Suppression of Il-8 Production in Response to InterferonGamma by Apoptosis-Specific eIF-5A siRNA

HT-29 (human colon adenocarcinoma) cells were transfected with siRNAdirected to apoptosis-specific eIF-5A. Approximately 48 hours aftertransfection the media was changed so that some of the test samples hadmedia with interferon gamma and some of the samples had media withoutinterferon gamma. 16 hours after interferon gamma addition, the cellswere washed, and the media, with or without TNF-alpha, was placed on thecells. The media (used for ELISA detection of IL-8) and the cell lysatewas harvested 8 or 24 hours later.

FIGS. 79 and 80 show that IL-8 is produced in response to TNF-alpha aswell as in response to interferon. Priming the cells with interferongamma prior to TNF treatment causes the cells to produce more IL-8 thaneither treatment alone. This may be due to the known upregulation of theTNF receptor 1 in response to interferon, so ‘priming the cells withinterferon allows them to respond to TNF better since the cells havemore receptors. siRNA against apoptosis-specific eIF-5A had no effect onIL-8 production in response to TNF alone (previous experiment) however,the siRNA blocked almost all IL-8 produced in response to interferon aswell as a significant amount of the IL-8 produced as a result of thecombined treatment of interferon and TNF. These results show that the byusing siRNAs directed against apoptosis-specific eIF-5A, the inventorshave the interferon signaling pathway leading to IL-8, but not the TNFpathway. FIG. 81 is a western showing up-regulation (4 fold at 8 hours)of apoptosis-specific eIF-5A in response to interferon gamma in HT-29cells.

Example 17 Human Lamina Cribrosa Culture

Paired human eyes were obtained within 48 hours post mortem from the EyeBank of Canada, Ontario Division. Optic nerve heads (with attached pole)were removed and placed in Dulbecco's modified Eagle's medium (DMEM)supplemented with antibiotic/antimycotic, glutamine, and 10% FBS for 3hours. The optic nerve head (ONH) button was retrieved from each tissuesample and minced with fine dissecting scissors into four small pieces.Explants were cultured in 12.5 cm² plastic culture flasks in DMEMmedium. Growth was observed within one month in viable explants. Oncethe cells reached 90% confluence, they were trypsinized and subjected todifferential subculturing to produce lamina cribrosa (LC) and astrocytecell populations. LC cells were enriched by subculture in 25 cm² flasksin DMEM supplemented with gentamycin, glutamine, and 10% FBS. Cells weremaintained and subcultured as per this protocol.

The identity and population purity of cells populations obtained bydifferential subculturing was characterized using differentialfluorescent antibody staining on 8 well culture slides. Cells were fixedin 10% formalin solution and washed three times with Dulbecco'sPhosphate Buffered Saline (DPBS). Following blocking with 2% nonfat milkin DPBS, antibodies were diluted in 1% BSA in DPBS and applied to thecells in 6 of the wells. The remaining two wells were treated with only1% bovine serum albumin (BSA) solution and only secondary antibody ascontrols. Cells were incubated with the primary antibodies for one hourat room temperature and then washed three times with DPBS. Appropriatesecondary antibodies were diluted in 1% BSA in DPBS, added to each welland incubated for 1 hour. Following washing with DPBS, the slide waswashed in water, air-dried, and overlayed with Fluoromount (VectorLaboratories). Immunofluorescent staining was viewed under a fluorescentmicroscope with appropriate filters and compared to the control wellsthat were not treated with primary antibody. All primary antibodies wereobtained from Sigma unless otherwise stated. All secondary antibodieswere purchased from Molecular Probes. Primary antibodies used toidentify LC cells were: anti-collagen I, anti-collagen IV, anti-laminin,anti-cellular fibronectin, anti-glial fibrillary acidic protein (GFAP),and anti-alpha-smooth muscle actin. Cell populations were determined tobe comprised of LC cells if they stained positively for collagen I,collagen IV, laminin, cellular fibronectin, alpha smooth muscle actinand negatively for glial fibrillary (GFAP). In this study, two sets ofhuman eyes were used to initiate cultures. LC cell lines #506 and #517were established from the optic nerve heads of and 83-year old male anda 17-year old male, respectively. All LC cell lines have been fullycharacterized and found to contain greater than 90% LC cells.

Treatment of LC Cells

Apoptosis was induced in lamina cribrosa cells using a combination of 50μM camptothecin (Sigma) and 10 ng/ml TNF-α (Leinco Technologies). Thecombination of camptothecin and TNF-α was found to be more effective atinducing apoptosis than either camptothecin or TNF-α alone.

Construction and Transfection of siRNAs

Small inhibitory RNAs (siRNAs) directed against human apoptosis-specificeIF-5A were used to specifically suppress expression of eIF5A in laminacribrosa cells. Six siRNAs were generated by in vitro transcriptionusing the Silencer™ siRNA Construction Kit (Ambion Inc.), Four siRNAswere generated against human apoptosis-specific eIF-5A (siRNAs #1 to#4). Two siRNAs were used as controls; an siRNA directed against GAPDHprovided in the kit, and an siRNA (siRNA #5), which had the reversesequence of the apoptosis-specific eIF-5A specific siRNA #1, but doesnot itself target eIF5A. The siRNAs were generated according to themanufacturer's protocol. The eIF5A and control siRNA targets had thefollowing sequences: siRNA #1 5′ AAAGGAATGACTTCCAGCTGA 3′ (SEQ ID NO:81); siRNA #2 5′ AAGATCGTCGAGATGTCTACT 3′ (SEQ ID NO: 82); siRNA #3 5′AAGGTCCATCTGGTTGGTATT 3′ (SEQ ID NO: 83); siRNA It 4 5′AAGCTGGACTCCTCCTACACA 3′ (SEQ ID NO: 84); siRNA #5′AAAGTCGACClTCAGTAAGGA 3′(SEQ ID NO: 85). Lamina cribrosa cells weretransfected with siRNA using LipofectAMINE 2000.

Lamina cribrosa cells were transfected when cell confluence was at 40 to70% and were generally seeded onto 8-well culture slides at 7500 cellsper well three days prior to transfection. Transfection mediumsufficient for one well of an 8-well culture slide was prepared bydiluting 25.5 proles of siRNA to a final volume of 21.2 μl in Opti-Mem(Sigma). 0.425 μl of Lipofectamine 2000 was diluted to a final volume of21.2 μl in Opti-Mem and incubated for 7 to 10 minutes at roomtemperature. The diluted Lipofectamine 2000 mixture was then added tothe diluted siRNA mixture and incubated together at room temperature for20 to 30 minutes, The cells were washed once with serum-free mediabefore adding 135 μl of serum-free media to the cells and overlaying42.4 μl of transfection medium. The cells were placed back in the growthchamber for 4 hours. After the incubation, 65 μl of serum-free mediaplus 30% FBS was added to the cells. Transfection of siRNA into cells tobe used for Western blot analysis were performed in 24-well plates usingthe same conditions as the transfections in 8-well slides except thatthe volumes were increased by 2.3 fold. Following transfection, laminacribrosa cells were incubated for 72 hours prior to treatment with 50 μMof camptothecin (Sigma) and 10 ng/ml of TNF-α (Leinco Technologies) toinduce apoptosis. Cell lysates were then harvested for Western blottingor the cells were examined for apoptosis

Detection of Apoptotic Cells

Transfected cells that had been treated with TNF-α and camptothecin for24 hours were stained with Hoescht 33258 in order to determine thepercentage of cells undergoing apoptosis. Briefly, cells were fixed witha 3:1 mixture of absolute methanol and glacial acetic acid and thenincubated with Hoescht stain (0.5 μg/ml Hoescht 33258 in PBS). After a10 minute incubation in the dark, the staining solution was discarded,the chambers separating the wells of the culture slide were removed, andthe slide was washed 3 times for 1 minute with deionized water. Afterwashing, a few drops of Mcllvaine's buffer (0.021 M citric acid, 0.058 MNa₂HPO₄.7H₂O; pH 5.6) was added to the cells and overlaid with acoverslip. The stained cells were viewed under a fluorescent microscopeusing a UV filter. Cells with brightly stained or fragmented nuclei werescored as apoptotic. A minimum of 200 cells were counted per well. TheDeadEnd™ Fluorometric TUNEL (Promega) was also used to detect the DNAfragmentation that is a characteristic feature of apoptotic cells.Following Hoescht staining, the culture slide was washed briefly withdistilled water, and further washed by immersing the slide twice for 5minutes in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, 8.1 mMNa₂HPO₄), blotting the slide on paper towel between washes. The cellswere permeabilized by immersing them in 0.2% Triton X-100 in PBS for 5minutes. The cells were then washed again by immersing the slide twicefor 5 minutes in PBS and blotting the slide on paper towel betweenwashes. 25 μl of equilibration buffer [200 mM potassium cacodylate (pH6.6), 25 mM. Tris-HCl (pH 6.6), 0.2 mM dithiothreitol, 0.25 mg/ml bovineserum albumin, and 2.5 mM cobalt chloride] was added per well andincubated for 5 to 10 minutes. During equilibration, 30 μA of reactionmixture was prepared for each well by mixing in a ratio of 45:5:1,respectively, equilibration buffer, nucleotide mix [50 μMfluorescein-12-dUTP, 100 μM dATP, 10 mM Tris-HCl (pH 7.6), and 1 mMEDTA], and terminal deoxynucleotidyl transferase enzyme (Tdt, 25 U/μl).After the incubation in equilibration buffer, 30 μl of reaction mixturewas added per well and overlayed with a coverslip. The reaction wasallowed to proceed in the dark at 37° C. for 1 hour. The reaction wasterminated by immersing the slide in 2×SSC [0.3 M NaCl, and 30 mM sodiumcitrate (pH 7.0)] and incubating for 15 minutes. The slide was thenwashed by immersion in PBS three times for 5 minutes. The PBS wasremoved by sponging around the wells with a Kim wipe, a drop of mountingmedia (Oncogene research project, JA1750-4 ML) was added to each well,and the slide was overlayed with a coverslip. The cells were viewedunder a fluorescent microscope using a UV filter (UV-G 365, filter set487902) in order to count the Hoescht-stained nuclei. Any cells withbrightly stained or fragmented nuclei were scored as apoptotic. Usingthe same field of view, the cells were then viewed using a fluoresceinfilter (Green H546, filter set 48915) and any nuclei fluorescing brightgreen were scored as apoptotic. The percentage of apoptotic cells in thefield of view was calculated by dividing the number of bright greennuclei counted using the fluorescein filter by the total number ofnuclei counted under the UV filter. A minimum of 200 cells were countedper well.

Protein Extraction and Western Blot Analysis

Protein was isolated for Western blotting from lamina cribrosa cellsgrowing on 24-well plates by washing the cells twice in PBS (8 g/L NaCl,0.2 g/L KCl, 1.44 g/L Na₂HPO₄, and 0.24 g/L KH₂PO₄) and then adding 50μl of lysis buffer [2% SDS, 50 mM Tris-HCl (pH 7.4)]. The cell lysatewas collected in a microcentrifuge tube, boiled for 5 minutes and storedat −20° C. until ready for use. Protein concentrations were determinedusing the Bicinchoninic Acid Kit (BCA; Sigma). For Western blotting, 5μg of total protein was separated on a 12 SDS-polyacrylamide gel. Theseparated proteins were transferred to a polyvinylidene difluoridemembrane. The membrane was then incubated for one hour in blockingsolution (5% skim milk powder, 0.02% sodium azide in PBS) and washedthree times for 15 minutes in PBS-T (PBS+0.05% Tween-20). The membranewas stored overnight in PBS-T at 4′C. After being warmed to roomtemperature the next day, the membrane was blocked for 30 seconds in 1μg/ml polyvinyl alcohol. The membrane was rinsed 5 times in deionizedwater and then blocked for 30 minutes in a solution of 5% milk in PBS.The primary antibody was preincubated for 30 minutes in a solution of 5%milk in PBS prior to incubation with the membrane. The primaryantibodies used were anti-eIF5A (BD Transduction Laboratories) at1:20,000 and anti-β-actin (Oncogene). The membranes were washed threetimes in PBS-T and incubated for 1 hour with the appropriateHRP-conjugated secondary antibodies diluted in 1% milk in PBS. The blotwas washed and the ECL Plus Western blotting detection kit (AmershamPharmacia Biotech) was used to detect the peroxidase-conjugated boundantibodies.

Results

Two lamina cribrosa (LC) cell lines were established from optic nerveheads obtained from male donors ranging in age from 83 years (#506) to17 years (#517). The cells isolated from the human lamina cribrosa hadthe same broad, flat morphology with prominent nucleus observed in otherstudies (Lambert et al., 2001). Consistent with the characterizations ofother groups, the LC cells showed immunoreactivity to alpha smoothmuscle actin (FIG. 82 a) as well as to a number of extracellular matrixproteins including cellular fibronectin (FIG. 82 b), laminin (FIG. 82c), collagen I, and collagen IV (data not shown) (Clark et al., 1995;Hernandez et al., 1998; Hernandez and Yang, 2000; Lambert et al.; 2001).Negative immunoreactivity of the LC cells to glial fibrillary acidicprotein (GFAP) was also observed consistent with previous findings (FIG.82 d) (Lambert et al., 2001). These findings support the identificationof the isolated cells as being LC cells rather than optic nerve headastrocytes.

Since TNF-α is believed to play an important role during theglaucomatous process, the susceptibility of LC cells to the cytotoxiceffects of TNF-α was examined. Confluent LC cells were exposed to eithercamptothecin, TNF-α, or a combination of camptothecin and TNF-α for 48hours (FIG. 83). Hoescht staining revealed that TNF-α alone was notcytotoxic to LC cells. Treatment with camptothecin resulted inapproximately 30% cell death of the LC cells. However, a synergisticincrease in apoptosis was observed when LC cells were treated with bothcamptothecin and TNF-α, a treatment which resulted in the death of 45%of LC cells by 48 hours. These results indicate that LC cells arecapable of responding to the cytotoxic effects of TNF-α when primed forapoptosis by camptothecin.

EIF5A is a nucleocytoplasmic shuttle protein known to be necessary forcell division and recently suggested to also be involved duringapoptosis. The expression of apoptosis-specific eIF-5A protein in LCcells being induced to undergo apoptosis by either camptothecin, orcamptothecin plus TNF-α. The expression of apoptosis-specific eIF-5A didnot alter significantly upon treatment with camptothecin except perhapsto decrease slightly (FIG. 84A). However, a significant upregulation ofapoptosis-specific eIF-5A protein was observed after 8 and 24 hours ofcamptothecin plus TNF-α treatment (FIG. 84B). These results indicatethat of apoptosis-specific eIF-5A expression is induced specifically byexposure TNF-α and expression correlates to the induction of apoptosis.This points to a role for apoptosis-specific eIF-5A in the apoptoticpathway downstream of TNF-α receptor binding.

In order to examine the importance of apoptosis-specific eIF-5Aexpression during TNF-α-induced apoptosis in LC cells, a series of foursiRNAs (siRNAs #1 to #4) targeting apoptosis-specific eIF-5A weredesigned and synthesized by in vitro transcription. To determine theeffectiveness of the siRNAs in suppressing apoptosis-specific eIF-5Aprotein expression, LC cell lines #506 and #517 were transfected witheach of the siRNAs and expression of apoptosis-specific eIF-5A proteinin the cell lysate was examined 72 hours later (FIG. 85). Forcomparison, cells were also transfected with either an siRNA againstGAPDH and/or a control siRNA (siRNA #5) having the same chemicalcomposition as siRNA #1 but which does not recognize apoptosis-specificeIF-5A. All siRNAs directed against apoptosis-specific eIF-5A werecapable of significantly suppressing apoptosis-specific eIF-5Aexpression in both LC cell lines (FIG. 85). The GAPDH siRNA was used asan additional control because, unlike the control siRNA #5 which simplyhas the reverse sequence of siRNA #1 and does not have a cellulartarget, it is an active siRNA capable of suppressing the expression ofit's target protein, GAPDH (data not shown). All four siRNAs againstapoptosis-specific eIF-5A were also capable of protecting transfected LCcells (#506) from apoptosis induced by 24 hour treatment with TNF-α andcamptothecin (FIG. 86). Using Hoescht staining to detect cell death, thesiRNAs (siRNAs #1 to #4) were found to be able to reduce apoptosis of LCcells by 59% (siRNA #1), 35% (siRNA #2), 50% (siRNA #3), and 69% (siRNA#4). Interestingly, the siRNA against GAPDH was also able to reduceapoptosis of LC cells by 42% (FIG. 86). GAPDH is known to have cellularfunctions outside of it's role as a glycolytic enzyme, including aproposed function during apoptosis of cerebellar neurons (Ishitani andChuang, 1996; Ishitani et al., 1996a; Ishitani et al., 1996b). In asimilar experiment we also demonstrated that siRNA #1 was able to reduceapoptosis of the LC line #517 by 53% in response to TNF-α andcamptothecin indicating that apoptosis-specific eIF-5A. siRNAs areprotective for LC cells isolated from different optic nerve heads (FIG.87). These results indicate that apoptosis-specific eIF-5A does have afunction during apoptosis and may be an important intermediate in thepathway leading to TNF-α-induced apoptosis in LC cells.

In order to confirm that LC cells exposed to TNF-a and camptothecin weredying by classical apoptosis, DNA fragmentation was evaluated in situusing the terminal deoxynucleotidyl transferase-mediateddUTP-digoxigenin nick end labeling (TUNEL) method. LC cells (#506) weretreated with TNF-α and camptothecin for 24 hours, 3 days aftertransfection with either an apoptosis-specific eIF-5A siRNA (siRNA #1)or a control siRNA (siRNA #5). The cells were also stained with Hoeschtto facilitate visualization of the nuclei. 46% of LC cells transfectedwith the control siRNA were positive for TUNEL staining while only 8% ofLC cells transfected with apoptosis-specific eIF-5A siRNA #1 werepositively labeled indicating that the apoptosis-specific eIF-5A siRNAprovided greater than 80% protection from apoptosis (FIG. 88). Similarresults were obtained with apoptosis-specific eIF-5A siRNA #4 whichprovided greater than 60% protection from apoptosis relative to thecontrol siRNA (data not shown).

Example 18 Blood Collection and Preparation of PBMCs

Approximately 10 ml of blood was collected from each healthy donor. Theblood was collected by venapuncture in a vacutainer containing sodiumcitrate as the anti-coagulant. The samples were processed within 24hours of collection.

A 60% SIP (9 parts v/v Percoll with 1 part v/v 1.5M NaCl) was cushionedon the bottom of 1.5 ml conical tubes. The blood was then layeredovertop with minimal mixing of the blood and Percoll cushion. Thesamples were centrifuged for 30 minutes total at 1000×g with slowacceleration in the first 5 minutes and slow deceleration in the last 5minutes. The pure serum at the very top of the resulting gradient wasremoved and the white cushion (1-2 ml) of PBMCs was collected and addeddropwise to a tube containing 10 ml of warm RPMI plus 15% FBS. The PBMCswere pelleted and counted.

Stimulation to Induce Cytokine Production in PBMCs Over a Time Course

PBMCs were isolated and seeded at 2×10⁵ to 5×10⁵ cells/well. The cellswere treated with phorbol 12-myristate 13-acetate (PMA; 100 ng/well). At72 hours the media was replaced and did not contain any stimulatingfactors. Then at 96 hours after PMA addition to PBMCs,lipopolysaccharide (LPS; 100 ng/well; from E. coli, serotype 0111) wasadded to the wells. Samples were collected before LPS addition (96 h),and at various times after addition as outlined in FIG. 91. Bothadherent cells (likely to be monocytes and macrophages) were collectedwith the floating cells (likely to be lymphocytes). To collect samplesfor analysis of cytokine secretion, the media from each well wastransferred to clean microcentrifuge tubes and cleared of any debris bycentrifugation at 13000×g for 3 minutes. The resulting pellet wascollected with the adherent cells. The media was stored at −20° C. in200-250 μl aliquots prior to analysis. The cells were washed with 1 mlof 37° C. phosphate buffered saline (PBS) and then lysed in boilinglysis buffer (50 mM Tris pH 7, 2% SDS; 100 μl per well). The celllysates were boiled and stored frozen at −20° C. The Western blot isshown in FIG. 92 and the corresponding ELISA in FIG. 93.

PBMC Stimulation to Induce Apoptosis-Specific eIF-5A Expression

PBMCs were collected and seeded at 2×10⁵ to 5×10⁵ cells/well. Todetermine which stimulators induce apoptosis-specific eIF-5A; as well asto see if they act synergistically, the PBMCs were stimulated withphytohemagglutinin (PHA; 100 ng/ml), phorbol 12-myristate 13-acetate(PMA; 100 ng/ml), lipopolysaccharide (LPS; 100 ng/ml) or all three (eachat 100 ng/ml). The samples were collected 12 and 36 hours afterstimulation and analyzed for apoptosis-specific eIF-5A expression (FIG.94).

Transfection of PBMGs

PBMCs were transfected the day they were prepared. Cells were seeded at2×10⁵ to 5×10⁵ cells/well (150 μl per well for transfection in a 24-wellplate). They were either transfected individually in each well (Donors77, 78 and 79; FIGS. 95 and 96) or all at once in a conical tube beforeseeding (Donors 80 and 84; FIG. 96). For each well of cells to betransfected, 15 pmoles of siRNA was diluted in 50 μl of Opti-MEM(Sigma). 1 μl of Lipofectamine 2000 (Invitrogen) was diluted in 49 μl ofOpti-MEM, incubated for 7 to 10 minutes, added to the diluted siRNA andincubated 25 minutes. The transfection medium was overlayed onto thecells were placed in the 37° C. growth chamber for 4 hours. The finaltransfection medium contained 9% serum. After the incubation, 250 μl ofserum-free RPMI+21% FBS was added to the cells to make the final serumconcentration 15%.

Stimulation to Induce Cytokine Production in PBMCs Post Transfection

72 hours after transfection of the PBMCs, as outlined above,lipopolysaccharide (LPS; 100 rig/well; from E. coli, serotype 0111) wasadded to the cells in 500 μl of media. The samples were collected at 24hours post stimulation. Both wells that were treated with LPS and wellsthat were transfected only (i.e. no stimulation) were collected. Tocollect samples for analysis of cytokine secretion, the media from eachwell was transferred to clean microcentrifuge tubes and cleared of anydebris by centrifugation at 13000×g for 3 minutes. The resulting pelletwas collected with the adherent cells. The media was stored at −20° C.in 200-250 μl aliquots prior to analysis. The cells were washed with 1ml of 37° C. phosphate buffered saline (PBS) and then lysed in boilinglysis buffer (50 mM Tris pH 7, 2% SDS; 1000 per well). The cell lysateswere boiled and stored frozen at −20° C. for BCA protein quantitation.

Example 19 Cell Culture

HT-29, a human colorectal adenocarcinoma cell line, was maintained inRPMI with 10% fetal bovine serum (FBS). U937, a histiocytic lymphomacell line, was grown in suspension in RPMI with 10% FBS. Both cell lineswere maintained in a humidified environment at 37° C. and 5% CO₂. Forexperiments with U937 cells, cells were counted and adjusted to 3×10⁵cells/ml two days before the start of the experiment. On the first dayof the experiment, cells were collected by centrifugation at 400×g for10 ruins, the cell pellet was resuspended in fresh RPMI media with 10%PBS, the centrifugation was repeated, and the repelleted cells wereresuspended in fresh RPMI media without PBS. The cells were counted andadjusted to 2×10⁶ cells/ml.

siRNA

siRNA sequences were designed based on the human apoptosis-specificeIF-5A sequence and were synthesized by Dharmacon RNA Technologies. Theapoptosis-specific eIF-5A siRNA (h5A1) target sequence was: 5′NNGCUGGACUCCUCCUACACA 3′. The corresponding double stranded siRNAsequence was:

5′ GCUGGACUCCUCCUACACAdTdT 3′ 3′ dTdTCGACCUGAGGAGGAUGUGU 5′The control siRNA (hcontrol) sequence was 5 NNACACAUCCUCCUCAGCUCG 3′.The corresponding double stranded siRNA sequence was:

5′ ACACAUCCUCCUCAGGUCGdTdT 3′ 3′ dTdTUGUGUAGGAGGAGUCCAGC 5′

Transfection of HT-29 Cells

The day before transfection, HT-29 cells were seeded at 105,000 cellsper well onto a 24-well plate. For each well of cells to be transfected,25.5 pmoles of siRNA was diluted in 50 μl of Opti-Mem (Sigma). 1 μl ofLipofectamine 2000 (Invitrogen) was diluted in 49 μl of Opti-Mem,incubated for 7 to 10 minutes and added to the diluted siRNA andincubated 25 minutes. The cells to be transfected were washed once withserum-free RPMI before adding 300 μl of serum-free RPMI and overlaying100 μl of transfection medium. The cells were placed hack in the growthchamber for 4 hours. After the incubation, 300 μl of serum-free RPMI+30%FBS was added to the cells.

Electroporation of U937 Cells

apoptosis-specific eIF-5A and control siRNA were diluted in Opti-Memmedia (Sigma). 400 μl cells (800,000 cells) and 100 pmoles siRNA weremixed in a 0.4 mm electroporation cuvette. The cells were electroporatedat 300 V, 10 mSec, 1 pulse with an ECM 830 Electrosquare porator (BTX,San Diego, Calif.). Following electroporation, the cells were gentlymixed and added to wells containing RPM and concentrated FBS so that thefinal FBS concentration was 10%.

Treatment of HT-29 Cells

TNF-α production was induced in HT-29 cells according to the methoddeveloped by Suzuki et al. 2003. HT-29 cells were primed with 200units/ml interferon gamma (Roche Diagnostics) 48 hours aftertransfection. After 16 hours of interferon gamma (IFNγ) priming thecells were washed with media and lipopolysaccharide (LPS; 100 ng/ml;from E. coli, serotype 0111; Sigma) was added at 100 μg/ml. After 8 or24 hours of LPS stimulation, the media from each well was transferred tomicrocentrifuge tubes and stored at −20° C. until assayed for TNFα byELISA. The cells were washed with 1 ml of phosphate buffered saline(PBS) heated to 37° C. and then lysed in boiling lysis buffer (50 mMTris pH 7, 2% SDS). The cell lysates were boiled and stored frozen at−20° C. The protein concentration in the cell lysates was determined bybicinchoninic acid assays (BCA) with bovine serum albumin used as thestandard.

IL-8 production was induced in HT-29 cells by treatment with IFNγ. HT-29cells were treated with 200 units/ml IFNγ 48 hours after transfection.After 24 hours of treatment, the media from each well was transferred tomicrocentrifuge tubes and stored at −20° C. until assayed for IL-8 byliquid-phase electrochemiluminescence (ECL). The cells were washed with1 ml of phosphate buffered saline (PBS) heated to 37° C. and then lysedin boiling lysis buffer (50 mM Tris pH 7, 2% SDS). The cell lysates wereboiled and stored frozen at −20° C. The protein concentration in thecell lysates was determined by bicinchoninic acid assays (BCA) withbovine serum albumin used as the standard.

Induction of Differentiation in U937 Cells

U937 cells were collected and counted 16 hours after electroporation.200,000 cells in 1 ml of media were added to each well of 24-wellplates. Macrophage differentiation was stimulated by adding phorbol12-myristate 13-acetate (PMA; 100 ng/ml). After 48 h with PMA., >80% ofthe monocytes had transformed from cells in suspension (monocytes) toadherent cells (macrophages). At 48 hours the media and any non-adherentcells were removed and fresh RPMI media with 10% FBS (1 ml per well) wasadded. The cells were left for 24 hours in fresh media to becomequiescent.

Stimulation to Induce Cytokine Production in U937 Cells

72 hours after PMA addition to U937 cells, lipopolysaccharide (LPS; 100ng/ml; from E. coli, serotype 0111), interferonγ (IFNγ; 100 Units/ml),or a combination of LPS and IFNγ were added to the wells. Samples werecollected before stimulator addition (72 h), and at various times afteraddition as outlined in FIG. 108. To collect samples for analysis ofcytokine secretion, the media from each well was transferred to cleanmicrocentrifuge tubes and cleared of any debris by centrifugation at13000×g for 3 mins. The media was stored at −20° C. in 200-250 ulaliquots prior to analysis. The cells were washed with 1 ml of 37″Cphosphate buffered saline (PBS) and then lysed in boiling lysis buffer(50 mM Tris pH 7, 2% SDS; 75 μl per well). Like wells were pooled. Thecell lysates were boiled and stored frozen at −20° C.

Cytokine Quantification

All media samples were stored frozen at −20° C. TNFα was quantifiedusing ELISA kits from Assay Designs according to the manufacturer'sinstructions with supplied standards for 0-250 pg TNFα/ml. For U937experiments media samples for TNFα were diluted 20 fold (0 h, 3 h LPS)or 80 fold (6 h, 24 h, 30 h LPS) with RPMI+10% FBS. IL-1β, IL-8, andIL-6 were quantified by liquid-phase electrochemiluminescence (ECL).Media from HT-29 experiments were not diluted. All cytokine measurementresults were corrected for the amount of total cellular protein (mg) perwell.

IL-8, IL-1β, and IL-6 were assayed by liquid-phase. Briefly, a purifiedmonoclonal mouse anti-mouse anti-human IL-8, IL-6 or IL-1 p (R & DSystems) were labeled with biotin (Igen, Inc., Gaithersburg, Md.). Inaddition, the goat anti-human IL-8, IL-6, or IL-1β antibody (R & D) werelabeled with ruthenium (Igen) according to the manufacturer'sinstructions. The biotinylated antibodies were diluted to a finalconcentration of 1 mg/mL in PBS, pH 7.4, containing 0.25% BSA, 0.5%Tween-20 and 0.01% azide, (ECL buffer). Per assay tube, 25 mL of thebiotinylated antibodies were pre-incubated at room temperature with 25mL of a 1 mg/mL solution of streptavidin-coated paramagnetic beads(Dynal Corp., Lake Success, N.Y.) for 30 min by vigorous shaking,Samples to be tested (25 mL) which had been diluted in RPMI or standardswere added to tubes followed by 25 ml, of ruthenylated antibody (finalconcentration 1 mg/mL, diluted in ECL buffer). The tubes were thenshaken for an additional 2 hours. The reaction was quenched by theaddition of 200 mL/tube of PBS and the amount of chemiluminescencedetermined using an Origen Analyzer (Igen).

SDS-PAGE and Western Blotting

The protein concentration in the cell lysates was determined bybicinchoninic acid assays (BCA) with bovine serum albumin used as thestandard. 5 μg of total cellular protein was separated by either 10% or14% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis). 10% gels were used for analysis of proteins above 50kDa (TLR4, IFNγ, TNF-R1, iNOS) while 14% gels were used forapoptosis-specific eIF-5A (17 kDa). Gels were transferred topolyvinylidene fluoride (PVDF) membranes with transfer buffer (48 mMTris, 39 mM glycine, 1.3 mM SDS, pH 9.2; 15V for 18 mins) using asemi-dry transfer unit (Bio-Rad). Membranes were blocked for 1 hour with5% skim milk in PBS-t (PBS with 0.1% Teen 20). Primary antibodies werediluted in the blocking solution and all blots were incubated at roomtemperature with shaking. Primary antibodies used wereapoptosis-specific eIF-5A (BD Biosciences; 1:20,000; incubate 1 hour;recognizes both apoptosis-specific eIF-5A and eIF5A2), TLR4 (Santa CruzBiotechnology Inc; TLR4 (H-80): sc-10741; 1:1000; incubate 2 hours),IFN-γRα (Santa Cruz Biotechnology Inc; IFN-γRα (C-20): sc-700; 1:1000;incubate 1 hour), TNF-R1 (Santa Cruz Biotechnology Inc; TNF-R1 (E1-5):se-8436; 1:200; incubate 3 hours), iNOS (BD Transduction Laboratories:61.0431; 1:10,000; incubate 1 hour) and β-actin (Oncogene; actin (Ab-1);1:20,000; incubate 1 hour). Following primary antibody incubations,blots were washed 3 times for 5-10 minutes with PBS-t. Horseradishperoxidase-conjugated (HRP) secondary antibodies were diluted in 1% skimmilk and incubated with the membrane for 1 hour. Secondary antibodiesused were anti-mouse IgG-HRP (Sigma; 1:5000; for apoptosis-specificeIF-5A and TNF-R1), anti-rabbit IgG-HRP (Amersham Pharmacia Biotech;1:2500; for TLR4 and IFNγ-Rα), anti-mouse IgM-HRP (Calbiochem; 1:5000;for actin). Following secondary antibody incubations, blots were washed4 times for 5-10 mins with PBS-t. Blots were developed with enhancedchemiluminescent detection reagent (ECL; Amersham Pharmacia Biotech)according to the manufacturers instructions and bands were visualized onX-ray film (Fuji).

RT-PCR

RT-PCR was performed according to Medvedev et al. 2002 in order toobserve changes in TLR4 mRNA expression in transfected HT-29 cells inresponse to IFNγ. Expression of GAPDH was used as a control to show thatequal amounts of cDNA were being used between samples. Increasing PCRcycles (20, 25, 30, and 35) were used to determine the optimal cyclenumber that resulted in detectable amplified products undernonsaturating conditions. PCR products were detected by ethidiumbromide—incorporation and were separated by agarose gel electrophoresis.RT-PCR of total mRNA isolated from siRNA-transfected HT-29 cells treatedwith or without IFNγ for 6 hours was used to detect TLR4 and GAPDHtranscripts. HT-29 cells were transfected with siRNA as described above.48 hours after transfection, the cells were treated with 200 units/mlIFNγ. Control cells which were not treated with IFNγ received only amedia change. Total mRNA was isolated using the GenElute Mammalian RNAminiprep kit (Sigma) according to the manufacturer's protocol foradherent cells. The media was removed and the cells were washed twicewith warm PBS. Lysis buffer was added to the cells and the lysate wastransferred to a microcentrifuge tube and total RNA was isolatedaccording to the manufacturer's protocol.

The primers for TLR4 (NM_(—)003266) were:

Forward 5′ CGGATGGCAACATTTAGAATTAGT 3′ Reverse 5′TGATTGAGACTGTAATCAAGAACC 3′ Expected fragment size: 674 bpThe primers for GAPDH (BC023632) were:

Forward 5′ CTGATGCCCCCATGTTCGTCAT 3′ Reverse 5′ CCACCACCCTGTTGCTGTAG 3′Expected fragment size: 599 bpThe total RNA was reverse transcribed using the following conditions:

Mix:

RNA 2.5 μg Poly (T) primer 6.25 μl Depc water to 13.75 μl Heat 70° C. 5min Chill on ice 5 min Add: 5× AMV Buffer 5.0 μl dNTPs (10 mM) 2.5 μlRnase Inhibitor 1.25 μl AMV RT 2.5 μl Heat 42° C. 60 min Heat 70° C. 10minA single PCR reaction was performed using the following conditions:

10X Tsg buffer 2.0 μl dNTP (10 mM) 0.4 μl forward primer (25 pmol/μl)0.4 μl reverse primer (25 pmol/μl) 0.4 μl MgCl₂ (15 mM) 2.0 μl cDNA 0.8μl H₂O 13.88 μl  Tsg polymerase 0.12 μl The PCR conditions for TI-124 were:

Heat to 95° C. 5 min

20, 25, 30, or 35 cycles of: 95° C. 1 min

-   -   55° C. 1 min    -   72° C. 2 min

Extend at 72° C. for 10 min Sink to 4° C.

The PCR conditions for OAPDH were:

Heat to 95° C. 5 min

20, 25, 30, or 35 cycles of: 95° C. 1 min

-   -   57° C. 1 min    -   72° C. 2 min

Extend at 72° C. for 10 min

Sink to 4° C.

1-14. (canceled)
 15. A method delivering siRNA to lunch cells of amammal in vivo, the method comprising mixing said siRNA with water anddelivering to a mammal intranasally.
 16. An siRNA of apoptosis-specificeIF-5A wherein said siRNA suppresses endogenous expression ofapoptosis-specific eIF-5A in a cell.
 17. The siRNA of claim 16, whereinapoptosis-specific eIF-5A is encoded by nucleotide sequence of SEQ IDNO:20.
 18. The siRNA of claim 16, wherein apoptosis-specific eIF-5A isencoded by nucleotide sequence of SEQ ID NO:41.
 19. the siRNA of claim16 wherein the siRNA has the sequence set forth in SEQ ID NO:30.
 20. ThesiRNA of claim 16 wherein the siRNA has the sequence set forth in SEQ IDNO:31.
 21. The siRNA of claim 16 wherein the siRNA has the sequence setforth in SEQ ID NO:32.
 22. The siRNA of claim 16 wherein the siRNA hasthe sequence set forth in SEQ ID NO:33.
 23. A method of inhibitingexpression of apoptosis-specific eIF-5A in a cell, the method comprisingadministering the siRNA of claim 16 to said cell whereby saidapoptosis-specific eIF-5A siRNA inhibits expression of endogenousapoptosis-specific eIF-5A in said cell.
 24. A method of inhibitingexpression of apoptosis-specific eIF-5A in a cell, the method comprisingadministering the siRNA of claim 19 to said cell whereby saidapoptosis-specific eIF-5A siRNA inhibits expression of endogenousapoptosis-specific eIF-5A in said cell.
 25. A method of inhibitionexpression of apoptosis-specific eIF-5A in a cell, the method comprisingadministering the siRNA to claim 16 to said cell whereby saidapoptosis-specific eIF-5A siRNA inhibits expression of endogenousapoptosis-specific eIF-5A in said cell.
 26. A method of inhibitingexpression of apoptosis-specific eIF-5A in a cell, the method comprisingadministering the siRNA of claim 19 to said cell whereby saidapoptosis-specific eIF-5A siRNA inhibits expression of endogenousapoptosis-specific eIF-5A in said cell.
 27. The method of claim 25wherein said inhibition of expression of endogenous apoptosis-specificeIF-5A in said cell has an effect on the cell selected from the groupconsisting of suppressing apoptosis in said cell, reducing expression ofp53 in said cell, reducing levels of cytokine produced in said cell,reducing levels of a cytokine produced in said cell, increasingexpression of Bcl-2 in said cell; reducing levels of myeloperoxidaseproduced in said cell, and reducing levels of active NFk beta in saidcell, reducing levels of TLR4 in said cell, reducing levels of TNFR-1 insaid cell and reducing levels of iNOS in said cell.
 28. The method ofclaim 26 wherein said inhibition of expression of endogenousapoptosis-specific eIF-5A in said cell has an effect on the cellselected from the group consisting of suppressing apoptosis in saidcell, reducing expression of p53 in said cell, reducing levels of acytokine produced in said cell, reducing levels of a cytokine producedin said cell, increasing expression of Bcl-2 in said cell; reducinglevels of myeloperoxidase produced in said cell, and reducing levels ofactive NFk beta in said cell, reducing levels of TLR4 in said cell,reducing levels of TNFR-1 in said cell and reducing levels of iNOS insaid cell.